JP2006104106A - Method for separating blood serum and method for culturing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To effectively utilize a PRP (platelet-rich plasma) and blood serum contained in blood, especially the blood of a patient oneself. <P>SOLUTION: There is provided a method for separating the blood serum 12 comprising charging an adsorptive member 9 composed of a glass material into a plasma 6 after separating the PRP and a material ingredient from the blood containing an anticoagulant and adsorbing and removing a fibrinogen 11 in the plasma 6. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for separating serum and a method for culturing cells.

  In recent years, it has become possible to regenerate the bone and repair the defect by replenishing the bone defect caused by bone tumor extraction, trauma, etc. with a bone grafting material. Hydroxyapatite (HAP) and tricalcium phosphate (TCP) are known as bone prosthetic materials. From the idea that no foreign matter is left in the body, for example, a calcium phosphate porous material such as β-TCP is used. Scaffolding material is used. When β-TCP is brought into contact with bone cells in the bone defect portion, so-called remodeling is performed in which osteoclasts absorb β-TCP and osteoblasts form new bone. That is, the bone prosthetic material compensated in the bone defect portion is replaced with the autologous bone over time (for example, see Non-Patent Document 1).

  In addition, in order to repair bone defects early, cultured bones that have been differentiated into osteoblasts by culturing mesenchymal stem cells attached to a bone grafting material in vitro are used. According to the cultured bone, it is possible to shorten the time until the bone forming action is started after the bone defect portion is filled, and the bone defect portion can be repaired at an early stage.

On the other hand, Patent Document 1 discloses that platelet-rich plasma (PRP) extracted from blood increases bone regeneration. Platelet-rich plasma contains various growth factors and growth factors. By using this for culture of cultured bone, cultured bone can be produced at an early stage. Moreover, since the components contained in the patient's own blood can be used, there is an advantage that there is no risk of infection due to an unknown infectious disease.
Uemura et al., “Tissue engineering in bone using biodegradable β-TCP porous material -Osferion, a new material that increases strength in vivo”, Medical Asahi, Asahi Shimbun, October 1, 2001, 30th Volume 10, No. 10, p. 46-49 JP 2003-55237 A

  To extract platelet-rich plasma from blood, the blood is extracted by centrifuging in two stages. At this time, fibrinogen (blood coagulation factor I) and prothrombin (blood coagulation factor II) contained in the blood are extracted. In order to prevent blood from coagulating due to the action of blood coagulation factors such as blood coagulation factor V and blood coagulation factor VIII, it is common to mix heparin or a citrate-based anticoagulant into the blood. It is.

  However, if an anticoagulant is mixed in the blood, platelet-rich plasma can be extracted without coagulating blood clotting factors, but the serum contained in the remaining plasma from which platelet-rich plasma has been extracted There is an inconvenience that it cannot be separated. That is, serum is separated from plasma by removing various blood coagulation factors contained in the plasma, but anticoagulants are mixed, so these blood coagulation factors are coagulated. It becomes difficult to make.

  Conversely, it is possible to coagulate blood clotting factors by adding a high concentration of coagulant, but in this case, the coagulant remains in the extracted serum, which is When used for culturing, insoluble crystals may precipitate in the medium during culturing, which may adversely affect the cultured cells. Therefore, conventionally, when platelet-rich crystals are extracted from blood, the remaining plasma has to be discarded, and there is a problem that the serum contained in the plasma cannot be effectively used.

  The present invention has been made in view of the above-described circumstances, and is a method for separating serum that makes it possible to effectively use platelet-rich plasma and serum contained in blood, particularly in the patient's own blood, and The object is to provide a method for culturing cells.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a serum separation method in which an adsorbing member made of a glass material is introduced into plasma after separating platelet-rich plasma and a formed component from blood containing an anticoagulant, and the fibrinogen in the plasma is adsorbed and removed. I will provide a.

  According to the present invention, the fibrinogen in plasma can be adsorbed to the adsorption member by introducing the adsorption member made of a glass material into the plasma. Since fibrinogen contains blood coagulation factors such as fibrin, serum can be easily separated by removing the adsorbing member that adsorbs these from the plasma. Therefore, serum can be separated from plasma containing an anticoagulant after the anticoagulant is introduced into the blood and the platelet-rich plasma and the formed component are separated and removed by centrifugation or the like. Can be used effectively without waste.

In the above invention, the adsorbing member may be made of glass beads.
By introducing glass beads into the plasma, the fibrinogen in the plasma adheres to the surface of the glass beads. For example, by removing the glass beads from the plasma with a filter, serum containing no blood coagulation factor can be easily obtained. Can be separated.

Moreover, in the said invention, an adsorption | suction member is good also as consisting of a glass rod.
By putting a glass rod into the plasma and stirring the plasma with the glass rod, the fibrinogen in the crystal can be adhered to the surface of the glass cap. And the serum which does not contain a blood coagulation factor can be easily isolate | separated by taking out a glass rod from plasma.

The present invention also provides a method in which magnetic beads having anti-blood coagulation factors adsorbed are put into plasma after separating platelet-rich plasma and formed components from blood containing an anticoagulant, and then magnetic beads are attached using a magnet. A method for separating collected serum is provided.
By reacting the anticoagulation factor adsorbed on the magnetic beads with the blood coagulation factor in plasma, the blood coagulation factor can be attached to the magnetic beads. Thereafter, serum can be easily separated by applying a magnet to the magnetic beads to be adsorbed and collected.

The present invention also provides a method for culturing cells in which platelet-rich plasma separated from blood containing an anticoagulant and serum separated by any of the separation methods described above are added to the medium and cultured.
According to the present invention, cells can be cultured using platelet-rich plasma and serum separated from the patient's own blood. Therefore, there is no risk of infection due to an unknown infectious disease, and cells can be cultured more safely and safely.

  According to the present invention, since serum can be extracted effectively from plasma after extracting platelet-rich plasma in blood, it is possible to effectively use serum that has been discarded so far. In particular, since platelet-rich plasma and serum contained in the patient's own blood can be used effectively, there is no risk of infection due to an unknown infectious disease, and the cells can be cultured more safely and securely.

A method for separating serum according to an embodiment of the present invention will be described below with reference to FIG.
The method for separating serum according to this embodiment is a method for separating serum from blood collected from a patient himself. Since blood collected from the patient himself is stored for a predetermined period after collection, it must be prevented from coagulating during storage. Therefore, for example, an anticoagulant is sealed in a container such as a blood transfusion bag, and when the collected blood is stored, the anticoagulant and blood are mixed, and the coagulation action by the blood coagulation factor in the blood is caused. It is suppressed.

  The present embodiment is a method for separating platelet-rich plasma and serum from blood treated so as not to coagulate by the action of an anticoagulant as described above. As shown in FIG. It includes a first step S1 for separating the minute, a second step S2 for separating the platelet-rich plasma from the remaining plasma, and a third step S3 for separating the serum from the plasma from which the platelet-rich plasma has been separated.

  The first step S1 for separating the formed component 2 from the blood 1 is performed by centrifuging the blood 1 at 2000 rpm for 4 minutes. The formed component 2 contains, for example, cells such as mesenchymal stem cells, red blood cells, and the like. By performing the first step S1, the formed component 2 in the blood 1 sinks to the bottom of the centrifuge tube 3 and the formed component 2 is separated by collecting the supernatant 4 as shown in FIG. Plasma 4 can be obtained. The formed component 2 may be returned to the patient as autologous blood.

  The second step S2 for separating the platelet-rich plasma 5 from the plasma 4 is performed by centrifuging the plasma 4 at 3300 rpm for 5 minutes. Thereby, as shown in FIG. 3, since the platelet-rich plasma 5 sinks to the bottom of the centrifuge tube 3, by collecting the supernatant 6 it is possible to obtain the plasma 6 separated from the platelet-rich plasma 5, and The platelet-rich plasma 5 remaining in the centrifuge tube 3 can be collected.

  Next, in a third step S3 for separating the serum 7 from the plasma 6 collected in the second step S2, a plurality of glass beads 9 are put into a container 8 containing the plasma 6 as shown in FIG. Is done. In the figure, reference numeral 10 denotes a vibration exciter that rotates or eccentrically rotates the container 8. By performing the third step S3, the fibrinogen 11, for example, fibrin, contained in the plasma 6 is adsorbed on the surface of the glass beads 9.

  Then, when the vibration is applied for about 10 minutes, the glass beads 9 to which the fiber base material 11 is attached are sunk in the bottom of the container 8. In this state, the supernatant 12 is removed by a suction nozzle (not shown) or the like Serum 12 can be collected by aspiration. In addition, after collecting the serum 12, the fibrinogen 11 remains in the container 8 in a state of adhering to the glass beads 9.

  In this case, according to the separation method according to this embodiment, an anticoagulant is mixed in the blood 1, and after separation and collection of the platelet-rich plasma 5, serum that cannot be separated by the usual separation method using a coagulant. 12 can be easily separated and recovered. On the other hand, unlike the serum separated and recovered by adding a high concentration coagulant, since the coagulant is not mixed in the serum 12, insoluble crystals are precipitated in the medium during the subsequent cell culture. It does not adversely affect cell culture.

  That is, according to the separation method according to the present embodiment, the platelet-rich crystal 5 and the serum 12 can be separated and collected from the blood 1, so that conventionally, the serum 12 and serum that were discarded when the platelet-rich crystal 5 was collected. When collecting 12, there is an advantage that the platelet-rich crystal 5 that has been discarded can be used effectively. In particular, when the blood 1 is autologous blood collected from the patient itself, it is possible to obtain autologous blood-derived platelet-rich crystals 5 and autoserum 12, which is an unknown infection as compared to the case of using other blood. There is an advantage that a safer and more healthy platelet-rich crystal 5 and serum 12 can be obtained without the risk of infection due to illness.

  Further, according to the culture method of culturing cells using these autologous blood-derived platelet-rich crystals 5 and autoserum 12 obtained by the separation method, there is no risk of infection due to unknown infectious diseases, as described above. There is an effect that it is possible to obtain cells that are grown safely and soundly.

  In the method for separating serum 12 in the above embodiment, the glass beads 9 are put into the container 8 and shaken, but instead, as shown in FIG. Alternatively, the glass rod 13 may be inserted into the container 8 containing the plasma 6 after being separated and recovered, and may be vibrated by the shaker 10. Alternatively, the glass rod 10 inserted into the plasma 6 stored in the container 8 may be agitated by rotating in the plasma 6.

  By doing in this way, as in the case of the above embodiment, the fiber base 11 present in the plasma 6 is adsorbed on the surface of the glass rod 13, so that the glass rod 13 is pulled out after stirring for a predetermined time. By leaving, the serum 12 is separated and collected in the container 8. As a result, the serum 12 can be easily recovered as in the case where the glass beads 9 are introduced, and the fibrinogen 11 can be removed from the serum 12 more easily than in the case of the glass beads 9. There are advantages.

Further, instead of using an adsorbing member made of a glass material having an adsorbing ability of the fiber base 11 such as the glass beads 5 and the glass rod 13 as shown in FIG. For example, the magnetic beads 15 adsorbed with anti-fibrinogen, anti-prothrombin, etc. may be put into the plasma 6 after separating and removing the platelet-rich plasma 5. The magnetic beads 15 are made of, for example, a magnetic material such as ferrite.
In this case, the magnetic beads 15 are put into the container 8 containing the plasma 6 and stirred for a predetermined time by the shaker 10, so that the anticoagulant factor 14 on the surface of the magnetic beads 15 and the blood in the plasma 6 are mixed. By reacting with the coagulation factor 11, the blood coagulation factor 11 can be coagulated and held on the magnetic beads 15.

Then, as shown in FIG. 6 (c), the magnetic beads 15 in the container 8 are arranged in an attracted state on the magnet 16 side by the magnetic force of the magnet 16 by bringing the magnet 16 close from the outside of the container 8. can do. At this time, the blood coagulation factor 11 attached to the magnetic beads 15 is also placed in an attracted state on the magnet 16 side.
Therefore, in this state, the serum 12 can be easily separated and collected by sucking the liquid 12 in the container 8 with a suction nozzle (not shown) or the like. By doing so, the blood coagulation factor 11 can be retained in the container 8 at the time of suction by the suction nozzle, so that the serum 12 can be collected more easily.
Further, by fixing the anticoagulant factor 14 on the surface of the magnetic beads 15, the anticoagulant factor 14 is prevented from being mixed into the collected serum 12.

  The cells to be cultured in the cell culturing method according to the present embodiment may be any cells, but in particular, bone marrow-derived stem cells, cord blood-derived stem cells, placenta-derived stem cells, and the like can be employed.

It is a flowchart which shows the separation method of the serum which concerns on one Embodiment of this invention. It is a figure explaining the 1st step in the separation method of FIG. It is a figure explaining the 2nd step in the separation method of FIG. It is a figure explaining the 3rd step in the separation method of FIG. It is a figure which shows the 1st modification of the 3rd step of FIG. It is a figure which shows the 2nd modification of the 3rd step of FIG.

Explanation of symbols

1 Blood 2 Component 5 Platelet-rich plasma 6 Plasma 9 Glass beads (adsorbing member)
11 Fibrinogen 12 Serum 13 Glass rod (adsorption member)
14 Anticoagulation factor 15 Magnetic beads (adsorption member)
16 magnets

Claims (5)

  A method for separating serum, wherein an adsorption member made of a glass material is introduced into plasma after separating platelet-rich plasma and formed components from blood containing an anticoagulant, and the fibrinogen in the plasma is adsorbed and removed.   The method for separating serum according to claim 1, wherein the adsorbing member comprises glass beads.   The method for separating serum according to claim 1, wherein the adsorbing member comprises a glass rod.   Method of separating serum by collecting magnetic beads using a magnet after introducing magnetic beads adsorbing anti-blood coagulation factor into plasma after separating platelet-rich plasma and formed components from blood containing anticoagulant .   A method for culturing cells in which platelet-rich plasma separated from blood containing an anticoagulant and serum separated by the separation method according to any one of claims 1 to 4 are added to a medium and cultured.

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