JP2005329222A6 - Serum preparation container and regenerative medicine method using the same - Google Patents

Abstract

A blood container suitable for producing a large amount of serum quickly and efficiently while ensuring high safety, a blood separation method using the same, and a regenerative medical method are provided.
In a blood component separation apparatus 1 for separating and storing collected blood into a plurality of blood components, the blood storage portion 10 for storing the blood, and the blood storage portion 10 in an aseptic and airtight manner. The blood reservoir 10 is provided with a function of generating serum to remove coagulation factors from the blood to such an extent that it can be used practically as serum. The component storage unit 20 stores each blood component generated by separating the blood in the blood storage unit 10.
[Selection] Figure 1

Description

  The present invention relates to a blood container, a blood separation method using the same, and a regenerative medicine method.

  Currently, in the field of regenerative medicine, research is being conducted to promote regeneration of a subject's tissue by proliferating or differentiating stem cells collected from the subject outside the body and then transplanting the stem cell into the subject. Stem cells have attracted attention as cells that have the pluripotency to differentiate into various tissues and organs and are the key to regenerative medicine.

  In vitro growth of stem cells is known to be effective if serum is added to the medium, but for the purpose of human treatment, use serum derived from animals other than humans. This should be avoided for safety reasons, and it is required to use serum prepared from blood derived from humans, particularly from subjects. Compared with blood tests, relatively more serum is required for stem cell culture in the field of regenerative medicine.

As a method for preparing such serum, Patent Document 1 discloses a method of using a blood collection tube containing a blood coagulation promoting individual made of glass powder, for example.
JP 2000-000228 A

  However, since the method described in Patent Document 1 uses a small-capacity blood collection tube for the purpose of testing blood, the preparation operation is repeated many times in order to prepare the necessary amount of serum for stem cell culture. It must be suitable for practical use.

  In addition, the prepared serum is often temporarily frozen or refrigerated until use. Therefore, it is necessary to transfer the blood collection tube to a storage container, and the possibility that the serum is contaminated with microorganisms increases by performing this operation many times. In addition, a serum separating agent is often added to such a blood collection tube, and the possibility that impurities are mixed into the serum from this serum separating agent cannot be denied. Therefore, it is not appropriate to use the method disclosed in Japanese Patent Application Laid-Open No. 2000-000228 in the field of regenerative medicine for humans from the viewpoint of safety and hygiene. Furthermore, with existing blood collection tubes for serum preparation, it is difficult to extract components other than serum due to the structure of the blood collection tube, and it cannot be reused because it is a blood clot. is there.

  As described above, it is desired to develop a serum preparation container suitable for preparing a large amount of serum in culturing stem cells.

  The present invention has been made in view of the above problems, and a blood storage container suitable for producing a large amount of serum quickly and efficiently while ensuring high safety, and blood using the same An object is to provide a separation method and a regenerative medicine method.

  In order to achieve the above-mentioned object, the present invention is provided with a serum generation function for generating an amount of serum that can be practically used by a coagulation activation action in the presence of a blood-derived liquid component and platelets. A blood component separation device is provided.

  Here, “blood” means whole blood composed of blood cells (red blood cells, white blood cells, platelets) and plasma (serum) as a liquid component, and a liquid containing at least one of these (for example, blood collected by component donation) ). “Serum” refers to a pale yellow liquid whose fluidity decreases when the collected blood is allowed to stand and then separated from a red clot (blood clot). “Serum” in the present invention is a cell whose production method is different from general serum in that it is not separated from clots, but the coagulation factor and growth factor contained therein are substantially the same as general serum. A liquid component in blood that is useful for culturing.

  The “blood-derived liquid component” refers to “a blood component other than blood cells” or “a mixed solution obtained by adding a drug such as an anticoagulant to a blood component other than blood cells”.

  “Coagulation activating action” refers to activating a coagulation factor in blood in order to remove the coagulation factor from blood.

  The “serum-producing function for producing a practically usable amount of serum” refers to an amount that can be used for stem cell culture, for example. In culturing stem cells, serum needs about 10% of the medium. Therefore, it is preferably 5 (ml) or more and 1000 (ml) or less, more preferably 10 (ml) or more and 600 (ml) or less.

  Specifically, the following are provided.

  The present invention is an apparatus for separating and storing collected blood into a plurality of blood components, and in the blood reservoir for storing the blood, an amount that can be practically used as serum is prepared. Therefore, a blood component separation device provided with a serum generation function for removing a coagulation factor from the blood is provided.

  According to the blood component separation device, a large amount of collected blood can be stored in the blood storage unit by providing the blood storage unit. Further, since the blood reservoir is provided with a serum generation function, platelets and coagulation factors in the blood can be rapidly activated. Furthermore, since these activators can be removed quickly, a large amount of serum can be prepared. The blood component separation device preferably includes a component storage unit that stores a plurality of blood components separated from blood, and the component storage unit and the blood storage unit are connected aseptically and airtightly. More preferably. As a result, a series of steps from blood collection to serum preparation can be performed without touching the outside air, so that the risk of contamination by microorganisms can be reduced.

  The blood component separation device according to the present invention can be used not only for human blood but also for blood of mammals such as rodents, livestock, primates, etc. It can also be used as a device.

  In the blood component separation apparatus, the plurality of blood components may include serum, and may include white blood cells and red blood cells.

  According to this invention, the serum separated by the blood component separation device is suitable for use in the field of regenerative medicine because it is a serum containing a large amount of cell growth factors. Furthermore, since the activator can be separately collected, the collected blood can be effectively used without being discarded.

  In the blood component separation device, the blood storage unit and the component storage unit are flexible bags, and the serum generation function is performed by a blood coagulation promoting individual disposed inside the blood storage unit. It may be given.

  The blood storage part and the component storage part are plastic bags, are lightweight and convenient to carry. Each of these is provided at least one, but a plurality of these may be provided, and it is preferable that one blood storage part and two or more component storage parts are provided. Each volume is not particularly limited as long as each blood component can be substantially separated from blood, but preferably 5 (ml) to 1000 (ml). ) To 600 (ml). Moreover, a serum production | generation function can be provided by arrange | positioning a blood coagulation acceleration | stimulation individual | organism | solid in the inclusion of this blood storage part. Blood coagulation-promoting individuals are contained to such an extent that blood coagulation factors such as fibrin and platelets can be removed from blood, and are insoluble in blood. The situation can be avoided.

  In the blood component separation apparatus, it is preferable that the blood coagulation promoting individual is insoluble in the blood and has a massive appearance. Further, it is more preferable that the movement is set in the blood stored in the blood storage unit.

  According to this invention, it becomes possible to improve the handling property at the time of manufacture by making a blood coagulation acceleration | stimulation individual | organism | solid into a granular form, a granular form, or a lump-like thing. Moreover, by setting the blood coagulation promoting individual to be movable, the contact with blood can be made smoother and the activation efficiency of blood can be improved.

  In the blood component separation apparatus, the specific gravity of the blood coagulation promoting individual may be larger than the blood components separated from the blood. Thereby, the prepared serum can be easily taken out from the blood reservoir.

  In addition, when preparing serum from blood, activation is performed after activation of activated factors such as platelets and blood coagulation factors, and centrifugation is performed. In this case, destruction of red blood cells (hemolysis) From the standpoint of preventing damage to the blood reservoir and the blood coagulation promoting individual, it is preferable that the appearance shape of the blood coagulation promoting individual is substantially spherical. For the purpose of rapid activation, the surface of the blood coagulation promoting material is preferably formed with a layer made of a silicon dioxide compound.

  As the silicon dioxide compound, at least one selected from glass, silica, diatomaceous earth, kaolin and the like can be used, but is not limited thereto.

  Moreover, if a magnet is used for the core of the blood coagulation promoter, blood can be stirred by applying a magnetic field to the blood reservoir, and the activated factor can be activated quickly.

  Since the blood coagulation promoting individual has a porous structure, the surface area per unit volume can be set large, which is preferable for activation. However, in this case, it is necessary to allow blood to enter the hole.

For blood coagulation promoting individuals in the blood reservoir, the surface area is set with a relationship of 0.1 (mm ² / ml) to 25 (mm ² / ml) with respect to the amount of blood that can be stored in the blood reservoir. It is preferable from the viewpoint of both promotion of activation and suppression of hemolysis. This value is the best condition value at the present time, and values other than this range also have the same effect are included in the present invention.

  In the blood component separation device, the blood reservoir may be one in which no serum separating agent is introduced.

  In the blood component separation device, at least two connection ports are formed in the blood reservoir, and one of the two connection ports is introduced for introducing the blood into the blood reservoir. The path may be hermetically connected, and on the other side, a lead-out path for deriving each blood component separated from the blood in the blood reservoir may be hermetically connected.

  According to this invention, it can protect from mixing of the blood component to serum by connecting the introduction path and the derivation | leading-out path to the connection port formed in the blood storage part.

  In the blood component separation device, the component storage portion includes two or more bags, and the lead-out path includes a plurality of lead-out rods connected to the bags, and at least one of the plurality of lead-out rods. The part may be shared.

  According to the present invention, the component accommodating portion is composed of two or more bags, and each bag is connected to one by using at least a part of the plurality of lead-out rods, thereby facilitating handling. In addition, microorganisms can be prevented from being mixed by keeping the respective parts airtight.

  In the blood component separation device, the blood reservoir may contain air. The air contained in the blood reservoir is preferably 0.03 (cc / ml) to 1 (cc / ml) with respect to the blood volume that can be stored.

  According to this invention, it is possible to obtain the same effect as when blood coagulation promoting individuals are contained by containing air in the blood reservoir.

  In addition, the blood separation method of the present invention provides a blood component separation that is provided with a serum-generating function that produces a practically usable amount of serum in the presence of blood-derived liquid components and platelets by clotting activation. The apparatus is characterized in that blood is separated into a liquid component and a non-liquid component using an apparatus.

  Specifically, the following method is provided.

  A device for separating and storing collected blood into a plurality of blood components, wherein the blood reservoir for storing the blood produces serum in an amount that can be practically used as serum A blood component separation device having a function, a storage step of storing collected blood in a blood storage unit, and a platelet in blood stored in the blood storage unit by activating the serum generation function And an activation promoting step that promotes activation of an activator including a coagulation factor, and a separation step that separates the activator that has been activated and aggregated in the activation promoting step from the blood. Separation method.

  In the blood separation method, the activation promoting step may be a step of shaking the blood component separation device.

  In the blood separation method, the serum generation function may be provided by a blood coagulation promoting individual arranged inside the blood reservoir.

  Further, in the blood separation method, each blood component derived in the deriving step may include serum.

  Further, in the blood separation method, the derivation step is a step of accommodating the blood component in the component accommodating portion by flowing a liquid component in each blood in a state where the blood coagulation promoting individual is fixed. There may be.

  Furthermore, in the blood separation method, the blood coagulation promoting individual in the derivation step may be fixed by a fixing means attached to at least one of the inside and the outside of the blood reservoir.

  Furthermore, in the blood separation method, the fixing means is one or more selected from the group consisting of a magnet, a clamp, a sandwiching body that sandwiches the blood reservoir, and a plurality of protrusions provided in the blood reservoir. It may be a thing.

  According to these inventions, it is possible to collect a large amount of blood at a time and quickly separate it. Furthermore, since it can be performed from the storage process to the introduction process without touching the outside air, there is little risk of contamination by microorganisms. Furthermore, you may provide the derivation | leading-out process which derive | leads out each said blood component except the said to-be-activated factor isolate | separated from the blood at the said isolation | separation process to the said component accommodating part.

  In the activation promoting step, the activator can be activated by shaking the whole blood component separation device or the blood reservoir. The shaking method is not particularly limited as long as it does not cause hemolysis, but it is preferable that the shaking speed is such that blood coagulation promoting individuals can move evenly in blood. When a blood coagulation promoting individual using a magnet for the core is used, the blood can be stirred by applying a magnetic field to the blood reservoir.

  In the separation step, fibrin is generated in a state where the activated activator is attached to the blood coagulation promoting individual. This facilitates serum recovery. In the derivation step, red blood cells can be collected more easily by fixing the blood coagulation promoting individual to which fibrin has adhered with fixing means.

  Furthermore, the present invention provides the following method for collecting fiber and regenerative medicine in blood.

  Liquid component and non-liquid using a blood component separation device provided with a serum generation function that generates a practically usable amount of serum by coagulation activation in the presence of blood-derived liquid component and platelets A method of separating fiber from the non-liquid component and separating the fiber from the non-liquid component.

  Using blood component separators that have a serum-generating function to generate serum in a quantity that can be used practically by coagulation activation in the presence of blood-derived liquid components and platelets, blood is converted into liquid components. It is separated into non-liquid components, the liquid components are added to the medium, cells collected from the subject are seeded and cultured on the medium, and the resulting cells or tissues are transplanted into the subject Regenerative medical method for use.

  In the regenerative medicine method, the liquid component may contain serum.

  Using blood component separators that have a serum-generating function to generate serum in a quantity that can be used practically by coagulation activation in the presence of blood-derived liquid components and platelets, blood is converted into liquid components. A regenerative medical method for transfusion to the subject after separation into a non-liquid component and adjusting the concentration of the non-liquid component.

  In the blood separation method, the non-liquid component may include serum, white blood cells, and red blood cells.

  Since these methods can be used to prepare a large amount of serum, it is possible to prepare an amount of autologous serum necessary for cell culture. In addition, even if blood transfusion is required for surgery, red blood cells and fibrin can be collected after serum collection, so the concentration can be adjusted for self-transfusion or fibrin can be used as a stem cell scaffold or wound barrier You can do it.

  As described above, the blood component separation device of the present invention is used to separate collected blood for each component, and includes a blood storage unit that stores blood, and the blood storage unit has a serum generation function. Therefore, it is possible to quickly prepare a large amount of serum in which the growth of microorganisms is suppressed, and it is suitable for preparing a large amount of serum used for stem cell culture in regenerative medicine. Furthermore, erythrocytes and other components after serum collection are suitable for use as blood for self-transfusion and as a barrier for wounds.

  Therefore, if the blood component separation device according to the present invention is used, a large amount of blood components including serum can be prepared (produced) quickly and efficiently from the collected blood while ensuring high safety. .

  Moreover, in the blood separation method of the present invention, the blood component separation device according to the present invention is used, and the storage step of storing the collected blood in the blood storage unit and the serum generation function are activated, An activation promoting step that promotes activation of activators including platelets and coagulation factors in blood stored in the blood reservoir, and the activated and aggregated activated in the activation promoting step A separation step of separating the factor from the blood, and if necessary, blood components remaining after collecting the serum are collected and used for autologous blood transfusion and stem cell scaffold or wound barrier.

  Therefore, if this serum preparation method is used, blood components with high safety can be produced in large quantities.

  Further, the regenerative medicine method of the present invention is obtained by adding serum prepared using the above blood separation method to a medium, seeding stem cells collected from a subject in this medium, culturing, and obtaining by culture. The cells or tissues to be treated were transplanted to the subject to be treated. In addition, if the patient has low circulating blood volume, such as children, or if a large amount of bleeding is a concern during transplantation, the collected red blood cells should be transfused. did.

  Therefore, if this regenerative medical method is used, it is possible to use a large amount of serum whose biological safety is ensured, and the tissue and function of the subject can be safely and reliably regenerated.

  The blood component separation device according to the present invention is a device capable of generating blood coagulation promoting function in a space for storing blood components for producing serum and generating a practically usable amount of serum.

  As a typical example of the device form, it is considered that at least one flexible bag is connected aseptically, liquid-tight, and air-tight and has a blood coagulation promoting function. It is done.

  Such a blood component distribution and storage device is similar in appearance to a so-called blood bag or separation bag, but the same configuration is adopted because these various components collected are aseptically and liquid-tight. This is because it has a long history of being a form suitable for airtight distribution.

  Moreover, the point which provided the blood coagulation promoting function is related to the essence of the present invention. Even if it is a mere flexible bag, when a liquid component containing a blood coagulation factor is accommodated, the coagulation factor is activated over time, and serum can be produced. However, not only is it relatively time consuming, but the growth factor contained in the serum is inactivated, so that the obtained activity is insufficient. Therefore, a blood coagulation promoting function was added. Thereby, in the said space, the liquid component containing a blood coagulation factor and the blood coagulation factor come into contact, and blood coagulation is promptly promoted. As a result, serum can be prepared in a very short time, and serum can be prepared in a state in which a decrease in activity is suppressed. This is true even when the bag itself is formed of a material having a blood coagulation promoting function, and the present invention does not exclude such a form. That is, generally, a form in which a storage space wall surface is formed of a material having a blood coagulation promoting function higher than that of a medical material used for a blood bag or a separation bag is also included in the scope of the present invention.

  As a substance having a blood coagulation promoting function, a glass processed body can be mentioned as one that has been confirmed to be most effective at present. Although it has long been known that glass has a blood coagulation effect, there has been no example of positive use for the production of a relatively large amount of serum. The inventors of the present invention used the processed glass as a substance having a function of promoting blood coagulation, made it exist in blood components that produce serum, and repeated contact with each other. It succeeded in generating without degrading.

  Further, air was used as a substance having a blood coagulation promoting function instead of the glass processed body. It is already known that blood will clot when it comes into contact with air, but none has been actively used to produce serum. Therefore, repeated contact with blood using air as a substance having a blood coagulation promoting function succeeded in producing a large amount of serum in a short time.

  It has been found that a substance having a blood coagulation promoting function is activated as the contact with blood increases. That is, it has been found that in the case of a glass processed body, it is effective to increase the specific surface area, and in the case of air, it is effective to increase the content. However, when a solid is used as a substance having a blood coagulation promoting function, such as a glass processed body, the concern about hemolysis must be considered.

  Next, the following forms can be considered as a form in which a liquid component containing a blood coagulation factor and a substance having a blood coagulation promoting function (hereinafter referred to as a blood coagulation promoting individual) are brought into contact.

  First, blood collected from a human body is stored in a blood storage part in which a blood coagulation promoting individual is stored, and blood coagulation is promoted to separate serum.

  Here, the separated serum is aseptically transferred to a bag other than the blood reservoir and can be stored in this bag. Alternatively, the separated serum can be aseptically led out and added to the cell culture system as a growth factor. In addition, other blood components (red blood cells, fibrin, etc.) separated simultaneously during serum separation can be used for blood transfusion and regenerative treatment.

  Secondly, the blood collected from the human body is stored in the blood reservoir, and after the blood component is separated in the state where the anticoagulant is added to the reservoir, the component that produces serum among the separated components It is conceivable that the liquid component containing blood coagulation factor and platelets are aseptically transferred to a separate bag body and serum is produced in the bag body. In this case, it is natural to store the above-mentioned blood coagulation promoting individual in the bag for generating serum, but a neutralizing agent for neutralizing the mixed anticoagulant is added. Thus, the function of the stored coagulation promoting individual is sufficiently exerted. Here, the separated serum is stored in the blood reservoir as it is or aseptically transferred to another bag and stored. The separated serum can be aseptically led out and added to the cell culture system as a growth factor. In addition, other blood components (red blood cells, fibrin, etc.) separated before and after serum separation can be used for blood transfusion and regenerative treatment.

  The third is a form related to the second form. In this embodiment, only the components that produce serum are collected from the blood before introduction into the device, and serum is produced in the same manner as in the second embodiment. In this form, so-called component blood is collected from the beginning, and when only the purpose of collecting serum and fibrin is intended, unnecessary blood components are immediately returned to the test subject. Physical burden is reduced.

Hereinafter, the present invention will be described in more detail.
<First Embodiment>
[Overall configuration of blood component separation apparatus 1]

  The configuration of the blood component separation device 1 according to the embodiment of the present invention will be described with reference to FIG. In FIG. 1, only the main part of the configuration of the blood component separation device 1 is extracted and illustrated.

  As shown in FIG. 1, the blood component separation device 1 according to the embodiment of the present invention is configured with a blood storage unit 10 and a component storage unit 20 as main elements. Among these, the blood reservoir 10 includes a body 11 formed in a bag shape by fusing two sheets of flexible resin material, for example, soft polyvinyl chloride, at the outer edge 11a. The glass processing body 12 is inserted into the main body 11. That is, the blood reservoir 10 has a configuration in which a glass processed body 12 as a blood coagulation promoting individual is accommodated inside a main body 11 as an exterior body.

  The blood reservoir 10 is similar to a so-called blood bag or separation bag in the internal volume and appearance of the main body 11, but is different from the blood bag in that the anticoagulant is not filled therein. It differs from a separation bag in that it contains a coagulation accelerator. Further, the inside of the blood reservoir 10 is sterilized in advance.

The glass processed body 12 in the main body 11 has, for example, a substantially spherical shape made of soda glass. Moreover, in FIG. 1, although it is set as the structure provided with the three glass processing bodies 12 in the blood storage part 10, when obtaining the blood coagulation promotion function, the surface area of the glass processing body 12 with respect to the blood volume which can be stored is set to 0. It is preferable to set with a relationship of 1 (mm ² / ml) or more.

  The glass processed body 12 is not joined to the inner wall or the like of the main body portion 11, and when the main body portion 11 is given a shaking action or a vibrating action, the glass processed body 12 is in a state in which it can float inside the main body section 11. Is set.

In addition, in order to prevent the red blood cells in the blood from being destroyed and causing hemolysis when the body part 11 is subjected to a shaking action or a vibration action after blood storage, or even when the blood is subjected to a centrifuge, It is preferable to set the glass processed body 12 with a surface area that has a relationship of 25.0 (mm ² / ml) or less with respect to the amount of blood that can be stored in the main body 11 of the blood reservoir 10. The reason why these numerical ranges are preferable will be described later.

  The component accommodating part 20 is comprised from the six bag bodies 21-26, and each consists of soft polyvinyl chloride. These have been sterilized beforehand.

  As shown in FIG. 1, two tubes 41 and 42 are airtightly connected to the upper edge of the main body 11 of the blood reservoir 10 at the connection port. Since the tube 41 plays a role of an introduction path for introducing blood, the blood collection needle 30 or a connection portion connectable to the blood collection needle is connected to the other end. The other tube 42 that is airtightly connected to the blood reservoir 10 is connected to the bags 21 to 26 via the tubes 43 to 46, 51 to 56 and the branch bodies 61 to 65. These play the role of a derivation path for deriving the separated blood components. These tubes 41 to 46 and 51 to 56 are made of a flexible resin material, for example, a material such as soft polyvinyl chloride. Here, the bag bodies 21 to 26 of the component storage unit 20 and the tubes 51 to 56 are also airtightly connected.

  Further, the sizes of the tubes 41 and 42 are set so that the inner diameter dimension thereof is smaller than the outer dimension dimension of the glass processed body 12. This is to prevent the glass processed body 12 from entering the tubes 41 and 42 when preparing and deriving serum.

  The blood reservoir 10 and the bags 21 to 26 and the tubes 41, 42, 51 to 56 are connected in a state where the internal space is isolated from the external environment. Moreover, also about each tube 42-46, 51-56, and each branch body 61-65, the area | region where the inside serum distribute | circulates is connected to the state isolated from the external environment. Specifically, they are connected by solvent adhesion, heat welding, ultrasonic welding or the like.

  Although not shown in FIG. 1, in the blood component separation apparatus 1 according to the embodiment of the present invention, blood and extracted serum are obtained by sandwiching required portions of the tubes 42 to 46 and 51 to 56 with clamps. When deriving, the flow path can be switched.

[About serum preparation]
A serum preparation operation using the blood component separation apparatus 1 having the above-described configuration will be described with reference to FIGS. Note that FIG. 2 is used in combination as appropriate for the explanation of the operation.

  As shown in FIG. 2, the blood separation operation using the blood component separation apparatus 1 is roughly composed of seven steps (S1 to S7).

  First, as a first stage of operation, blood is collected by inserting the blood collection needle 30 in FIG. 1 into the subject (patient). At this time, the blood collected from the blood collection needle 30 is stored in the blood storage unit 10 located below via the tube 41 (storage step S1). Here, a breakable partition wall is provided between the tube 42 and the blood storage unit 10 so that blood collected in the blood storage unit 10 does not flow into the component storage unit 20. Alternatively, the path of the tube 42 is closed on the base side of the blood reservoir 10 using a clamp or the like. The storage step S1 is completed after collecting a required amount in consideration of the physical condition of the patient at the time of blood collection. The required amount here is about 200 to 600 (ml) when there is no problem in the patient's physique and physical condition.

  In collecting blood, a blood collecting machine widely used for donating blood can be used.

  Next, as shown in FIG. 2, after the storage step S1 is started, the blood storage unit 10 is shaken so as to be parallel thereto (activation promotion step S2). As shown in FIG. 3, the blood storage unit 10 storing the collected blood is gently stirred by the shaking device 100 and comes into contact with the glass processed body 12 housed therein. Then, platelets and coagulation factors contained in blood coagulate on the surface of the glass processed body 12, and growth factors derived from these are released from the platelets activated during coagulation. (In addition, if this activation promoting step is performed at a low temperature, it is effective for promoting platelet aggregation.)

  In addition, since the blood reservoir 10 is formed in the same outer dimensions as a normal blood bag, a known shaking device can be used for shaking the blood reservoir 10. Furthermore, although not shown in FIG. 3, since the blood reservoir 10 is connected to the bags 21 to 26 via the tubes 41 to 46 and 51 to 56, they can be folded and shaken. It is.

  After the storage step S1, the blood collection needle 30 is removed from the blood collection target, a part of the tube 41 connecting the blood collection needle 30 and the blood storage unit 10 is melted, and at the same time, the melted end is welded (melting) Step S3). The tube 41 can be melted using a fusing machine 110 (so-called sealer) as shown in FIG.

  On the other hand, the blood reservoir 10 separated from the patient undergoes the activation promotion step S2 together with the component storage unit 20 and the tubes 42 to 46, 51 to 56, and further to the branch bodies 61 to 65 connecting them. Then, it is put together in a compact and subjected to a centrifuge (centrifugation step S4). At this time, the tube 42 is maintained in a state in which the path is closed by a breakable partition wall or a clamp as in the storage step S1.

The fusing step S3 and the centrifugation step S4 may be performed before the activation promotion step S2 when blood is collected by adding an anticoagulant in advance. In this case, centrifugation is performed under the following conditions.
Centrifugation of whole blood: 4,400 g × 4-6 (min.), 2,250 g × 10 (min.)
Centrifugation of platelet rich plasma (PRP): 1,100 g × 4-6 (min.)

  Centrifugation conditions for the blood reservoir 10 are set according to the amount of stored blood and the types of components to be separated, and are set to, for example, 2250 (g) × 10 (min.), 4 (° C.). Is done. The blood reservoir 10 after centrifugation will be described with reference to FIG.

  As shown in FIG. 5, in the blood reservoir 10 that has been centrifuged after passing through the activation promoting step S2, it is roughly divided into three layers of serum 71, leukocytes 72, and red blood cells 73, depending on the centrifugation conditions. . Further, the glass processing body 12 has a platelet and a coagulation factor 74 coagulated on the surface thereof attached to the bottom of the main body portion 11a of the blood reservoir 10 (hereinafter referred to as “coagulated body-attached glass processing body 14”). Is sinking in. As shown in the enlarged portion of the figure, the coagulated body-attached glass processed body 14 is obtained by attaching a coagulated body 74 in which platelets and coagulation factors are coagulated to the surface of the glass processed body 12. Here, in order to distinguish the presence or absence of adhesion of the solidified body, reference numeral 14 is attached to the glass processed body after the solidified body 74 is adhered.

  As described above, the serum 71, which is the supernatant component in the state of FIG. 5, contains growth factors derived from these sufficiently released from platelets and coagulation factors in the activation promotion step S2. Also in the centrifugation step S4, the two tubes 41 and 42 that are hermetically connected to the upper edge of the main body 11 of the blood reservoir 10 are both in a closed state. It is isolated and microorganisms do not enter.

  Returning to FIG. 2, the activatable factors activated from the activation promotion step S2 to the centrifugation step S4 are aggregated and separated from the blood (separation step S5).

  In addition, the serum 71 separated and extracted in the blood reservoir 10 in the separation step S5 is subdivided in order with respect to all or part of the bag bodies 21 to 26 in the component storage unit 20 (derivation step S6). The derivation method will be described with reference to FIG.

  As shown in FIG. 6, when the extracted serum 71 is to be led out to the bag body 21 of the component storage unit 20, the path of the tube 43 is closed using the clamp 90, and blood is stored in this state. The blood reservoir 10 is pressurized (F1) with the pressurizer 80 installed outside the unit 10. As described above, the tube 41 that is airtightly connected to the blood storage unit 10 is melted at the middle 41a at the time when the storage step S1 is finished, and at the same time, the end and the vicinity 41b are welded.

  Therefore, by receiving the pressurization F1, a part of the serum 71, which is the supernatant extracted by the separation, is led to the serum storage bag 21 through the tube 42, the branch body 61, and the tube 51. .

  In addition, about the path | route closure of the tube 43, it implements by pinching the tube 43 which has a flexibility between the disc 91 and the base 92 of the clamp 90. FIG.

  Returning to FIG. 2, after the bag body 21 is filled with a required amount of serum 71, the tube 51 is melted and welded (melting step S <b> 7). As shown in FIG. 7, the fusing and welding are performed using the same method as that for fusing and welding the tube 42 before the centrifugation step S4. Further, as shown in FIG. 8, the bag body 21 filled with the serum 71 is subjected to a storage treatment such as frozen storage.

  The derivation step S6 and the fusing step S7 are sequentially performed on each of the bags 21 to 26, and when all or part of the bags 21 to 26 are filled with the serum 71, the serum preparation operation is performed. finish. Furthermore, if necessary, the red blood cells 73 can be washed and diluted with an anticoagulant such as physiological saline, CPD, ACD-A solution, or a blood storage solution such as MAP, and stored as blood for transfusion. This method will be described later.

[Advantages of blood component separation device 1]
In the blood component separation device 1 according to the embodiment of the present invention, a blood reservoir 10 is constituted by a main body 11 having an internal volume equivalent to that of a blood bag, and a glass processed body 12 disposed therein, Since serum is prepared from blood inside the blood reservoir 10, a larger amount of serum can be prepared at a time than in the case where a conventional blood collection tube is used, which is effective in terms of the process in the preparation work. . This also reduces the risk of the prepared serum being contaminated by microorganisms and the like, and is suitable for preparing highly safe serum.

  In addition, since the blood component separating apparatus 1 contains the glass processed body 12 as a blood coagulation promoting individual, blood clots adhere to the surface of the glass processed body 12 during serum preparation, and the serum is separated when serum is collected. Prevents fibrin and blood clots from entering.

  In addition to the above effects, the blood component separation device 1 is hermetically connected in a state where the blood reservoir 10 and the bags 21 to 26, the tubes 41, 42, 51 to 56, and the internal space are isolated from the external environment. Therefore, blood or serum is not exposed to the external environment. Therefore, further safety is ensured.

  Moreover, since the said tubes 41, 42, and 51-56 have flexibility and are formed from the material which can be cut and welded, when separating these paths | routes timely, an inside becomes an external environment. There is no exposure. Therefore, it can be said from this aspect that the blood component separation device 1 has high safety.

  Generally, when blood is stored in a container that does not have an anticoagulant (for example, ACD-A solution) inside, if the container is left for 20 (min.) Or more after blood collection, platelets in the blood gradually The coagulation factor coagulates, but particularly when the amount of air in the container is small, blood coagulation is not promoted, which causes a problem that it takes a long time to coagulate.

  On the other hand, in the blood component separation device 1 according to the embodiment of the present invention, the glass processed body 12 as a blood coagulation promoting individual having a blood coagulation promoting function is inserted into the blood reservoir 10 that stores blood. Therefore, most platelets (for example, 95% or more of platelets) and coagulation factors are coagulated within 10 (min.), And the serum preparation operation can be performed rapidly. Therefore, serum can be rapidly prepared by using the blood component separation device 1 according to the embodiment of the present invention. Further, since most of the red blood cells remaining in the blood component separation apparatus 1 after the serum is prepared through the activation promoting step S2 described above, the red blood cells may be reused as a component for transfusion. .

  Further, for example, in the field of regenerative medicine, a lot of highly safe serum is required for culturing stem cells. However, if the serum 71 is prepared and stored using the blood component separation device 1, it can be safely and quickly. Serum can be prepared and stored. Therefore, by using the blood component separation device 1 according to the present embodiment, it is possible to perform regenerative treatment of tissue or function with high efficiency while ensuring high safety for a patient.

Moreover, in the said embodiment, when the glass processing body 12 is set with the surface area which has the relationship of 0.1 (mm < ² > / ml) or more with respect to the blood volume which can be stored in the blood storage part 10. FIG. Is more promoted in the activation of platelets and coagulation factors in the blood. Further, when the surface area of the glass processed body 12 with respect to the storable blood volume is set to be within the range of 0.1 (mm ² / ml) or more and 25.0 (mm ² / ml) or less, the activity It is possible to achieve both suppression of hemolysis and promotion of activation of platelets and coagulation factors in the activation step S2 and the centrifugation step S4.

  In addition, although the number of the glass processed bodies 12 in the blood reservoir 10 is three in this embodiment, it is not limited to this. It is reasonable that the number of the glass processed bodies 12 to be accommodated is actually 1 or more and 50 or less.

  In the blood component separation apparatus 1, the glass processed body 12 is used as a blood coagulation promoting individual, but the blood coagulation promoting individual is not limited to this. For example, it is also possible to use a structure in which the contact area with blood is composed of an inorganic substance selected from at least one selected from silicon dioxide compounds such as silica, diatomaceous earth, and kaolin. Further, the substance disposed in the contact region is not limited to an inorganic substance.

  Further, in the blood component separation apparatus 1, the blood reservoir 10 has a blood coagulation promoting function by accommodating a blood coagulation promoting individual (glass processed body 12) having a blood coagulation promoting function in the blood reservoir 10. However, even if the blood coagulation promoting individual is not necessarily housed in the blood reservoir 10, the blood coagulation promoting function may be achieved. For example, a part of the inner wall of the main body part 11 of the blood storing part 10 may be covered with the inorganic substance.

  Moreover, about the glass processing body 12 in the said blood component separation apparatus 1, the external shape was made into the substantially spherical shape, However, The external appearance shape of the blood coagulation acceleration | stimulation individual | organism | solid which concerns on this invention is not limited to this. However, it is preferable to form the outer surface (contact region with blood) with a continuous curved surface from the viewpoint of suppressing the generation of hemolysis and the tearing of the bag during centrifugation.

  Further, in the derivation step S6, as shown in FIG. 9, platelets are adhered to the surface of the glass processed body by blocking the glass processed body to which fibrin has adhered, for example, from the outside of the blood storing section 10 with a clamping section 16 such as a clamp. Then, the clotting factor can be separated and red blood cells can be derived. In addition, it is preferable that the contact surface with the blood reservoir 10 has a corrugated shape or an uneven shape so that erythrocytes, fibrin washing liquid, and the like can circulate even after the blood reservoir 10 is sandwiched.

  In the embodiment of the present invention, the solid glass processed body 12 is used. However, if a substance having a blood coagulation promoting function is formed on the outer surface, the solid glass processed body 12 is not necessarily solid. For example, a porous structure may be used, and the entire region in contact with blood including the wall surface in the hole may be coated with a silicon dioxide compound such as glass.

Second Embodiment
The blood component separation device according to the present embodiment is different from the blood component separation device 1 according to the first embodiment described above in the form of a glass processed body housed in the blood reservoir 10. Therefore, below, the form of a glass processed body is demonstrated using FIG. 10, and description about another part is abbreviate | omitted.

  As shown in FIG. 10, the solidified body-attached glass processed body 14 according to the present embodiment is the same as the glass processed body 12 in that it has a substantially spherical appearance, but the core body portion 141 and the surface layer portion 142. It is characterized by having a two-layer structure consisting of

  Of the two layers constituting the solidified body-attached glass processed body 14, the core body portion 141 is composed of a magnet. On the other hand, the surface layer part 142 is comprised from soda glass as an inorganic substance same as the material which comprised the said glass processed body 12, for example.

  In the case of using the blood reservoir 10 in which the coagulated body-attached glass processed body 14 having such a structure is housed, the blood reservoir 10 is shaken and a magnetic field during the activation promoting step S2 in FIG. By acting, it is possible to obtain an effect that blood agitation is promoted. That is, by applying a magnetic field to the blood reservoir 10 from the outside with, for example, a stirrer such as a magnetic stirrer, the coagulated body-attached glass processed body 14 causes a rotational motion or the like in the container. Contact with blood with high efficiency.

  Therefore, in the blood component separation device provided with the coagulated body-attached glass processed body 14 in the blood reservoir 10, it is possible to activate platelets and coagulation factors more quickly than the blood component separation device 1, and more quickly. Serum can be prepared.

  Moreover, when the glass processed body 14 is comprised with the magnet, it can fix with a magnet from the outer side of the blood storage part 10 in derivation | leading-out process S6.

<Third Embodiment>
The blood component separation device according to this embodiment is different from the blood component separation device 1 according to the first embodiment described above in that one of the bag bodies 21 to 26 constituting the component storage unit 20 is used for air bleeding. (See FIG. 1). At the time of blood collection, only the volume of the tube 41 is inevitably mixed into the blood reservoir 10, but it is preferable that each blood component is not present during the separation step S5. Therefore, if the air vent bag according to the present embodiment is installed between the blood reservoir 10 and the component container 20, it is possible to remove only air from each blood component before the separation step S5. In addition, since the other structure in this embodiment is the same as that of 1st Embodiment, description about another part is abbreviate | omitted.

<Fourth embodiment>
As shown in FIG. 11, the blood component separation device according to the present embodiment contains air 15 instead of the blood coagulation promoting individual added to the blood reservoir 10 of the blood component separation device 1 according to the first embodiment. There are some differences. In this case, it is not necessary to add a blood coagulation promoting individual in advance, which leads to a reduction in manufacturing cost. The content of the air 15 is preferably 0.03 (cc / ml) to 1 (cc / ml) with respect to the blood volume that can be stored, and the leakage of air that has been sealed in advance so as to reach the content is used. It is preferable that the tube 41 has a mechanism for preventing the time.

  Air and blood coagulation promoting individuals may be used in combination.

<Fifth Embodiment>
As shown in FIG. 12, the blood component separation device according to the present embodiment is a glass processed body in at least one of the bag bodies 21 to 26 that constitute the component storage portion of the blood component separation device 1 according to the first embodiment. There is a difference when 12 (blood coagulation promoting individual) is added. In the present embodiment, a citrate neutralizing agent containing calcium ions may be further added to the container to which the glass processed body 12 is added. In this case, the blood reservoir 10 may be a so-called “blood donation blood bag” to which an anticoagulant such as a CPD solution is added.

  The blood in the blood reservoir 10 is separated to some extent by centrifugation or the like, but since an anticoagulant is added, serum cannot be produced as it is. In this embodiment, the blood in the blood reservoir 10 is neutralized with the neutralizing agent added in the bag body 21 to activate the growth factor in the blood, and the generated serum is led to the bag body 22. It becomes possible.

<Sixth Embodiment>
As shown in FIG. 13, the blood component separation device according to the present embodiment contains air 15 instead of the blood coagulation promoting individual added to the bag body 21 of the blood component separation device 1 according to the fifth embodiment. There are differences. In this case, as in the fourth embodiment, it is not necessary to add a blood coagulation promoting individual in advance, leading to a reduction in manufacturing cost. The content of the air 15 is preferably 0.03 (cc / ml) to 1 (cc / ml) with respect to the blood volume that can be stored, and the leakage of air that has been sealed in advance so as to reach the content is used. It is preferable that the tube 51 has a mechanism for preventing the time.

  Air and blood coagulation promoting individuals may be used in combination.

  Moreover, in the blood component separation device 1 according to the first embodiment and the fourth embodiment, the component storage portion is composed of the six bag bodies 21 to 26. However, the number of bags constituting the component storage portion is not limited to this. You are not limited to this. Furthermore, in the blood component separation apparatus 1 according to the first embodiment and the fourth embodiment, a glass processed body is used as a blood coagulation promoting individual, but the same applies to a case where air is contained instead of the glass processed body. The effect of. In this case, it is preferable that it is 0.03 (cc / ml) to 1 (cc / ml) with respect to the blood volume which can be stored.

  Further, the present invention can effectively use red blood cells and fibrin remaining in the blood reservoir 10 after separating blood. This will be described in detail below.

<Seventh embodiment>
In the first embodiment, a blood component separation device 1 that separates blood is used. As shown in FIG. 14, blood is separated and blood is extracted into the bag 21 (component storage unit), and then the blood storage unit 10 has a coagulant-attached glass processed body 14 to which fibrin adheres and residues such as red blood cells. Remains. A physiological saline is previously placed in any one of the component storage units 21 to 26 that are airtightly connected to the blood storage unit 10 in which serum is not stored, or physiological saline is placed in the tube 41 of the blood storage unit 10. The saline-containing bag 120 containing water can be connected and mixed with the red blood cells in the blood reservoir 10, and then used as blood for transfusion (see FIG. 15). Further, the coagulated body-attached glass processed body 14 to which fibrin is adhered can be further washed after all blood components are derived, and the fibrin obtained after washing can be used as a scaffold for stem cells or a barrier for wounds.

  Furthermore, in 1st Embodiment and 2nd Embodiment, although the structure which connected between the blood storage part and the component accommodating part with the tube etc. was employ | adopted as a blood component separation apparatus, the part and blood serum which preserve | save blood are preserve | saved It is not always necessary to configure the portion to be performed as a separate container. For example, it is possible to prepare a single container that can be partially cut off by fusing, welding, etc., store blood in the container, and use it as the component storage unit 20 from now on. Further, in the derivation step S6, a clamp that sandwiches the blood reservoir 10 so as not to derive the glass processed body 12 to which fibrin has adhered or the coagulated body-adhered glass processed body 14 has been mentioned, but is not limited thereto. Absent.

  Below, the result of having examined the effectiveness of the serum prepared using the blood component separation device in the first embodiment, the effectiveness of the collected red blood cells, and the proliferation of the stem cells will be described. In the experiment, an exterior body made of polyvinyl chloride was used.

[Example 1]
[Confirmation of activation promotion effect]
A large and small glass processed body made of soda glass was added to the blood container under the conditions shown in Table 1. 20 ml of fresh human blood was placed in this blood container, incubated with stirring, and 1.5 ml was collected after 10, 20, 30, 60, and 90 minutes, and the platelet count was measured. For stirring, a stirring (shaking) device (Multi-shaker MMS-300 manufactured by Tokyo Science Instruments) was used, and for blood cell counting, a blood cell counting device (multi-item automatic blood cell counter K-4500, manufactured by Sysmex) was used.

  FIG. 16 shows the relationship between the number of platelets and the incubation time. From this, it can be seen that the larger the surface area ratio of the glass processed body per 1 ml of blood is, the more activated platelets are aggregated. Moreover, it was shown that the sample to which the glass processed body was not added required a considerable time of about 90 minutes until blood aggregation. Furthermore, even if it is allowed to stand for a long time without adding a glass processed body and promotes the release of growth factors from platelets, the activation of other coagulation is insufficient. Large amounts of fibrin often precipitate. Therefore, in the containers of Samples B to I according to the experimental examples, platelets can be rapidly aggregated by containing the glass processed body, and the platelet-derived growth factor required for serum preparation is highly efficient. It can be seen that it can be released.

[Example 2]
[Confirmation of growth factor release effect]
The growth factor release effect was examined. Five samples are selected from the nine samples used in Example 1, and fresh blood of five subjects is added to each sample, and growth factors are measured after 20 minutes. Specifically, after incubation in the same manner as in Example 1 under the conditions shown in Table 2 below, the amount of growth factor (TGF-β1, PDGF-BB) was measured with a commercially available test kit (R & D SYSTEMS). It measured using the reader | leader (Multiskan BICHROMATIC Labsystem company make), and expressed as ratio with the quantity in the test substance of the contact area 0 with a glass processed body. The results are shown in FIGS. In the figure, the horizontal axis represents the glass surface area, and the vertical axis represents the amount of each growth factor. From FIG. 17, when the glass processed body was added as little as 0.6 mm ² per 1 ml of blood, the amount of growth factor released was greatly increased. However, it was found that when the surface area ratio was extremely increased, the amount of TGF-β1 was almost balanced. Moreover, in FIG. 18, the increase of the growth factor was confirmed only by adding a glass processed body slightly. Furthermore, it was found that when the surface area ratio was extremely increased as in FIG. 17, the PDGF-BB amount was almost balanced.

Example 3
[Examination of hemolysis by glass processing body]
From Examples 1 and 2, it was confirmed that the addition of the processed glass body was effective in platelet activation and growth factor increase. However, there was concern that hemolysis may be caused during serum preparation due to the addition of the glass processed body. Therefore, the relationship between the amount of glass processed body added and hemolysis was examined.

Samples A, F, H, I used in Example 1 and bovine blood prepared in each sample of J to M prepared in a wide range of glass surface area ratios per ml of blood were placed and incubated while stirring. This was sampled over time, and the hemoglobin concentration of the centrifuged supernatant was measured (hemoglobin B-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.). The results after 90 minutes incubation are shown in FIG. It can be seen that the increase rate of the supernatant hemoglobin increases as the surface area of the glass processed body accommodated in the blood reservoir increases. This indicates that the glass processed body has an excessive contact area with blood, and causes a lot of destruction of red blood cells (hemolysis) in the activation promotion step or the centrifugation step. However, up to 12.5 mm ² was confirmed to show hemolysis equivalent to that of Sample A to which no glass processed body was added.

Example 4
[Confirmation of rat stem cell proliferation]
In the blood reservoir in which the contact area of the glass processed body per 1 ml of blood is 0 (mm ² ) or 1.5 (mm ² ), and blood collected from a human is stored in any of the glass processed bodies Shake for 20 minutes. After shaking, the blood was centrifuged (centrifugation conditions: 2250 (g) × 10 (min.), 4 (° C.)), and the supernatant was separated. The supernatant was heat treated at 56 ° C. for 30 minutes, filtered through a 0.22 (μm) filter, and stored frozen at −80 ° C. The supernatant was thawed during cell culture and added to the cell culture medium. Cells obtained from rat femur bone marrow were cultured in advance for 7 days, and the obtained adherent cells were used as bone marrow-derived cells for this example. Seed 10,000 cells per well and add 10% concentration of the glass processed body to the culture medium, supernatant obtained without added glass processed body, or commercially available cell culture Fetal calf serum was added and cultured. The number of cells was counted 1, 3, 7 days after the start of culture. The results of confirming the effect on cell proliferation are shown in FIG.

  As shown in FIG. 20, it can be seen that the presence or absence of contact with the glass processed body during the preparation of serum to be added when culturing rat bone marrow-derived cells significantly affects cell proliferation. In addition, the supernatant obtained by adding the glass processed body gave better results than the fetal bovine serum generally used so far.

Example 5
[Recovering red blood cells]
The collected red blood cells were confirmed. The contact area of the glass processed body was 12.5 (mm ² ) per 1 ml of blood, and after shaking 20 (ml), it was shaken for 60 minutes with stirring on a multi shaker MMS-300 (manufactured by Tokyo Science Instruments Co., Ltd.) . The change in the number of red blood cells over time was examined using blood collected in a test tube and allowed to stand for the same time by a conventional serum preparation method. The result is shown in FIG. From this, when the number of red blood cells immediately after blood collection was 100%, about 80% of the red blood cells remained after shaking for 60 minutes with the glass processed body. On the other hand, when serum was collected by the conventional method, since most of the blood in the container became blood clot, only 10% of red blood cells were collected 60 minutes after blood collection.

Example 6
[Serum recovery from platelet rich plasma (PRP)]
It was confirmed that serum containing a large amount of growth factors could be prepared from blood collected in advance using an anticoagulant such as a CPD solution or platelet-rich plasma (PRP) prepared by blood donation. Fresh human blood to which CPD was added was prepared so that the final concentration was 12.2%. The CPD-added blood was centrifuged at 760 g for 10 minutes (22 ° C.) to prepare platelet-rich plasma. As shown in Table 4, the obtained platelet-rich plasma 0.8 mL was incubated at 37 ° C. in a container to which calcium chloride and a glass processed body had been added in advance, and shaken appropriately. After the platelet-rich plasma was added, the time until fibrin precipitated from the platelet-rich plasma and the fluidity decreased in appearance was measured. Each specimen with reduced fluidity was immediately centrifuged under conditions of 2,250 g and 10 minutes (4 ° C.), and the resulting supernatant was separated. Thereafter, the amount of PDGF-BB and the amount of TGF-β1 contained in the supernatant were measured. The measured amount of each growth factor is shown in FIG. 22 as a ratio (%) to the amount of each growth factor contained in the serum prepared from the same blood.

  As shown in FIG. 22, by adding the addition amount of the glass processed body and increasing the area, the clotting time of platelet-rich plasma was shortened, and the release amounts of PDGF-BB and TGF-β1 were also increased.

Example 7
[Confirmation of air addition effect]
Air or a glass processed body was added to the blood container under the conditions shown in Table 5, respectively. 20 (ml) of human fresh blood is put into this blood container and incubated with stirring on a multi shaker MMS-300 (manufactured by Tokyo Science Instruments Co., Ltd.), and the platelet count after 20 minutes is determined by a multi-item automatic blood cell counter K. -4500 (manufactured by Sysmex Corporation) was used for counting.

  As shown in FIG. 23, in Sample 1 to which neither a glass processed body nor air was added, nearly 90% of platelets remained even after 20 minutes. When only air was added (specimen names 2 to 4), the residual rate of platelets decreased in proportion to the amount of air added. Further, when the glass processed body and air were combined, the decrease in platelet residual rate was remarkable, and a proportional relationship was observed between the amount of air added and the platelet residual rate. Therefore, although not as much as the glass processed body, air alone has an effect of promoting platelet activation and aggregation, and further, when combined with the glass processed body, an effect of further promoting platelet activation and aggregation can be obtained. It was suggested.

  From Example 1 described above, it was found that in order to rapidly aggregate platelets, it is effective to accommodate a glass processed body as a blood coagulation promoting individual in the blood reservoir. That is, if a blood reservoir containing a glass processed body is used, it is possible to quickly and efficiently generate platelet-derived growth factors that are necessary for preparing serum from collected blood. Therefore, if such a glass processed body is stored in the blood component separation apparatus, a large amount of serum can be rapidly prepared (produced).

  In addition, from Example 2, serum prepared from blood collected in a container containing a glass processed body has sufficiently released growth factors useful for cell growth in the preparation process, and as a result, It was confirmed that the serum contributed to the growth of bone marrow-derived cells by adding it to the medium. Therefore, storing the glass processed body in the blood component separator is useful for preparing serum effective for promoting cell growth.

  Further, from Example 3, from the viewpoint of the amount of supernatant free hemoglobin in the serum separated by the centrifugation step, the excessive glass processed body is accommodated in the blood reservoir to destroy red blood cells during shaking and centrifugation. (Hemolysis) will be caused and it turns out that it is not desirable.

  Further, after preparing serum that effectively acts on cell culture from Examples 4 to 7, most of the red blood cells could be recovered as individual isolated cells instead of clots. This suggests that autologous blood can be preserved as blood for transfusion at the time of stem cell transplantation in patients with low circulating blood volume or patients undergoing transplantation that may be accompanied by a large amount of bleeding. Therefore, components other than serum can be collected by preparing serum under shaking using a blood collection container containing a glass processed body.

As described above, when considering Examples 1 to 7, the glass processed body in the blood reservoir is used for the amount of blood that can be stored in the container from the viewpoint of promoting the activation of platelets and coagulation factors and collecting the growth factors. It is desirable to set the surface area of the glass processed body to have a relationship of 0.1 (mm ² / ml) or more, and when considering the factor of hemolysis, a relationship of 25.0 (mm ² / ml) or less It can be seen that it is preferable to set so as to have

  As described above, the blood component separation device of the present invention can quickly and in large quantities prepare serum in which the growth of microorganisms is suppressed. Therefore, it is suitable for preparing a large amount of serum used for stem cell culture in regenerative medicine.

It is the figure which showed the blood component separation apparatus which concerns on 1st Embodiment of this invention. It is a figure which shows the procedure from blood collection by the blood component separation apparatus which concerns on 1st Embodiment of this invention to serum preservation | save. It is the figure which is shaking the blood storage part 10 of the blood component separation apparatus which concerns on 1st Embodiment of this invention. It is the figure which is made to melt | fuse the tube 41 of the blood storage part 10 of the blood component separation apparatus which concerns on 1st Embodiment of this invention. It is sectional drawing which showed the blood storage part 10 after centrifuging the blood component separation apparatus which concerns on 1st Embodiment of this invention. It is sectional drawing which shows the method to derive | lead-out the serum 71 prepared in the blood storage part 10 of the blood component separation apparatus which concerns on 1st Embodiment of this invention to the bag body 21 for serum preservation | save. It is the figure which has melted the tube 42 of the blood storage part 10 and the bag of the blood component separation device according to the first embodiment of the present invention. It is the figure which showed the bag body 21 which passed through the fusing process in the blood component separation apparatus which concerns on 1st Embodiment of this invention. It is the perspective view which showed the blood storage part 10 in the derivation | leading-out process after centrifuging the blood component separation apparatus which concerns on 1st Embodiment of this invention. It is a perspective view (partial sectional view) showing a coagulation body adhesion glass processing object 14 concerning a blood ingredient separation device concerning a 2nd embodiment of the present invention. It is the figure which showed the blood component separation apparatus 1 which concerns on 4th Embodiment of this invention. It is the figure which showed the blood component separation apparatus 1 which concerns on 5th Embodiment of this invention. It is the figure which showed the blood component separation apparatus 1 which concerns on 6th Embodiment of this invention. It is the figure which showed the blood component separation apparatus 1 which concerns on 7th Embodiment of this invention. It is the figure which showed the process which introduce | transduces the physiological saline into the blood storage part of the serum preparation apparatus which concerns on 5th Embodiment of this invention. FIG. 6 is a characteristic diagram showing the relationship between the elapsed time from blood collection and the residual rate of platelets according to Example 1. It is a characteristic view which shows the relationship between the area | region of the glass processed body which contacts the blood which concerns on Example 2, and the release amount to the serum of cell growth factor (TGF- (beta) 1). It is a characteristic view which shows the relationship between the area | region of the glass processed body which contacts the blood which concerns on Example 2, and the release amount in the serum of a cell growth factor (PDGF-BB). It is a characteristic view which shows the relationship between the area of the glass processing body which contacts the blood which concerns on Example 3, and the amount of supernatant free hemoglobin. It is the figure which showed the relationship between each sample which concerns on Example 4, and the number of cells. It is the figure which showed the time-dependent change of the collection rate of the red blood cell of each sample which concerns on Example 5. FIG. It is the figure which showed the release rate of the growth factor of each specimen which concerns on Example 6, and the coagulation time of platelet-rich plasma. It is the figure which showed the platelet residual rate of each test substance which concerns on Example 7. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Blood component separation apparatus 10 Blood storage part 110 Fusing machine 120 Saline containing bag body 20 Component accommodating part 21-26 Bag body 30 Blood collection needle 11 Main body part 11a Outer edge part 12 Glass processed body 14 Coagulated body adhesion glass processed body 141 Core Body part 142 Surface layer part 16 Clamping part 20 Component storage part 21 Bag body 30 Blood collection needle 41-46, 51-56 Tube 61 Branch body 71 Serum 72 Leukocyte 73 Red blood cell 74 Coagulation body 80 Pressurizer 90 Clamp 91 Disc 92 Base 100 Shaking apparatus

Claims (22)

  A blood component separation apparatus provided with a serum generation function for generating a practically usable amount of serum in the presence of blood-derived liquid components and platelets by a coagulation activating action.   2. A fluid comprising the blood-derived liquid component and the platelets is stored in the same space, and serum production is promoted by contacting a blood coagulation promoting individual present in the fluid. The blood component separation device according to 1.   3. The blood according to claim 1 or 2, wherein blood that has not been substantially separated is stored, the blood coagulation promoting individual is present in the blood, and contact with the blood promotes serum production. Blood component separation device.   A component composed of blood-derived liquid component and platelets separated from the blood in advance is stored, and the blood coagulation promoting individual exists in the same space where these are stored, and a neutralizing agent for the anticoagulant is added. The blood component separation device according to claim 1 or 2, wherein serum production is promoted by adding and contacting with blood.   Blood that has not been substantially separated is stored, blood-derived liquid components separated from the blood and platelets are further stored, and the blood coagulation is promoted in the same space in which these are stored. The blood component separation apparatus according to claim 1 or 2, wherein an individual is present, and serum production is promoted by adding a neutralizing agent for an anticoagulant and contacting with the blood.   6. The blood component separation device according to claim 2, wherein the blood coagulation promoting individual is insoluble in the blood.   The blood component separation device according to any one of claims 2 to 6, wherein the blood coagulation promoting individual has a massive appearance.   The blood component separation device according to any one of claims 2 to 7, wherein the blood coagulation promoting individual is set so as to be able to move in the blood stored in the blood storage unit.   The blood component separation device according to any one of claims 2 to 8, wherein the specific gravity of the blood coagulation promoting individual is larger than each of the blood components separated from the blood.   The blood component separation device according to any one of claims 2 to 9, wherein the outer surface of the blood coagulation promoting individual includes an inorganic layer.   The blood component separation device according to claim 10, wherein the inorganic substance includes a silicon dioxide compound.   The blood component separation device according to any one of claims 2 to 11, wherein the blood coagulation promoting individual includes a magnet in a core.   The blood component separation device according to any one of claims 2 to 12, wherein the blood coagulation promoting individual has a porous structure having pores into which the blood can enter.   The blood component separation device according to any one of claims 2 to 13, wherein the blood coagulation promoting individual is agitated and contacted with a fluid composed of the blood-derived liquid component and the platelets.   The blood component separation and storage device according to any one of claims 1 to 14, comprising a plurality of containers for storing the produced serum.   A blood component separation device that is provided with a serum generation function that generates a practically usable amount of serum by coagulation activation in the presence of blood-derived liquid components and platelets, and this blood component separation device is sterile A blood component separation and storage device having storage means connected to each other.   Using blood component separators that have a serum-generating function to generate serum in a quantity that can be used practically by coagulation activation in the presence of blood-derived liquid components and platelets, blood is converted into liquid components. A blood separation method for separation into non-liquid components.   Liquid component and non-liquid using a blood component separation device provided with a serum generation function that generates a practically usable amount of serum by coagulation activation in the presence of blood-derived liquid component and platelets A method of separating a non-cellular component from the non-liquid component by separating into a non-liquid component.   Using blood component separators that have a serum-generating function to generate serum in a quantity that can be used practically by coagulation activation in the presence of blood-derived liquid components and platelets, blood is converted into liquid components. It is separated into non-liquid components, the liquid components are added to the medium, cells collected from the subject are seeded and cultured on the medium, and the resulting cells or tissues are transplanted into the subject Regenerative medical method for use.   The blood separation method according to claim 17, wherein the liquid component includes serum.   Using blood component separators that have a serum-generating function to generate serum in a quantity that can be used practically by coagulation activation in the presence of blood-derived liquid components and platelets, blood is converted into liquid components. A regenerative medical method for transfusion to the subject after separation into a non-liquid component and adjusting the concentration of the non-liquid component.   The blood separation method according to claim 19, wherein the non-liquid component includes white blood cells and red blood cells.

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