KR20080113297A - Apparatus and process for perparing human serum for cell culture - Google Patents

Abstract

It is intended to provide a serum which contains a large amount of a growth factor capable of efficiently promoting the growth of stem cells. Use is made of a human serum for cell culture which shows a remaining platelet content within 20 minutes after the blood collection of 20% or lower based on the total platelet content and the release rate of a cell growth factor of 20% or higher. ® KIPO & WIPO 2009

Description

Apparatus and process for producing human serum for cell culture {Apparatus and process for perparing human serum for cell culture}

The present invention relates to an apparatus and method for producing human serum for cell culture, and more particularly, to an apparatus and method for producing human serum for cell culture containing a large amount of cell proliferation factors.

This application claims the benefit of priority based on Japanese Patent Application No. 2004-335344, filed November 19, 2004, the contents of which are incorporated herein by reference.

Currently, in the field of regenerative medicine, studies have been conducted to promote tissue regeneration of a subject by transplanting stem cells collected from the subject in vitro and then transplanting them into the subject. Stem cells have a multipotent ability to differentiate into various tissues and organs, and are attracting attention as cells that hold the key to regenerative medicine.

In vitro culture of stem cells is known to be effective when serum is added to the medium.However, for the purpose of human treatment, the use of serum derived from animals other than humans should be avoided for safety reasons. It is required to use a serum prepared from blood derived from, in particular, the subject. In comparison with blood tests, relatively more serum is required for the culture of stem cells in the field of regenerative medicine.

As a method of preparing such a serum, the method of using the blood collection tube which accommodated the blood coagulation | solidification solid which consists of glass powder, for example is disclosed (refer patent document 1). In addition, in order to rapidly separate coagulants such as fibrin incorporated into serum, a method of easily recovering serum containing a large amount of growth factors by contacting free powder and blood is disclosed (Patent Document 2). Reference). Moreover, the method of manufacturing using human plasma as a raw material is disclosed (refer patent document 3). Moreover, the method of obtaining a growth factor by adding a calcium compound and glass beads to plasma is disclosed (refer patent document 4).

Patent Document 1: Japanese Patent Application Laid-Open No. 2000-000228

Patent Document 2: Japanese Patent Application Laid-Open No. 4-83165

Patent Document 3: Japanese Patent Application Laid-Open No. 2001-275662

Patent Document 4: Japanese Patent Publication No. 2004-269409

However, the methods disclosed in Patent Literatures 1 and 2 use a small blood collection tube for the purpose of testing blood, and thus, a preparation operation is performed several times in order to prepare an amount of serum necessary for culturing stem cells. It is not suitable for practical use because it must be performed. Moreover, since the method disclosed by patent document 3 uses thrombin as an anticoagulant, it is unpreferable in the point that the risk of infection by using a biological origin product remains. In addition, in the method described in Patent Document 4, since the plasma containing only a small amount of the cell growth factor is used as a raw material, the obtained serum does not contain sufficient cell growth factor, so that stem cells cannot be cultured efficiently. .

In view of the above problems, it is an object of the present invention to provide a serum containing a large number of growth factors that can efficiently promote the proliferation of stem cells.

More specifically, the present invention provides the following.

(1) Human serum for cell culture obtained from a fluid containing blood-containing liquid components and platelets containing a blood coagulation factor, the platelet residual rate within 20 minutes of blood collection exceeds 0% and 20% of the total platelet volume A cell culture serum having a release rate of cell proliferation factor of 20% or more and 100% or less.

According to the invention of (1), it is possible to promptly promote the release of cell proliferation factors by setting the platelet survival rate within 20 minutes of blood collection to be more than 0% and 20% or less of the total platelet amount. In addition, it is possible to efficiently propagate stem cells by setting the cell growth factor release rate to 20% or more and 100% or less. In addition, by using a fluid containing at least blood-derived nuclear components and platelets, it is possible to produce a serum containing more growth factors than the amount prepared from the plasma, thereby producing a serum having a cell proliferation effect equivalent to that of fetal bovine serum. It can manufacture.

Here, "plasma" refers to whole blood consisting of blood cells (red blood cells, white blood cells, platelets) and plasma (serum), which is a liquid component, and a liquid containing at least one of them (for example, blood collected by component blood donation). Say). In addition, "serum" means the pale yellow liquid which fluidity falls when the collected blood is left to stand and isolate | separates from a red coagulation mass (blood clot) after that. The term "serum" in the present invention refers to a liquid component in blood which is useful for cell culture in which a coagulation factor and a growth factor contained therein are substantially the same as a general serum, although the production method is different from a general serum in that it is not separated from blood clots. Say The "blood-derived liquid component" refers to a mixed solution in which a drug such as an anticoagulant is added to blood components other than blood cells or blood components other than blood cells. In addition, "cell growth factor" means platelet growth factor (PDGF), transforming growth factor (TGF-β1), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), brain Derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF) and the like.

The release rate of the cell growth factor is defined as the latent amount (100% release) of the cell growth factor in serum prepared from a predetermined amount of blood collected in a vacuum blood vessel, and the ratio of the cell growth factor to the latent amount is determined. Say.

(2) Human serum for cell culture obtained from a fluid containing a blood-derived liquid component and a platelet containing a blood coagulation factor, wherein the content of the cell growth factor is greater than that of human serum prepared from plasma. .

According to the invention of (2), it is possible to efficiently propagate stem cells by increasing the content of the cell proliferation factor of the human serum according to the present invention more than when prepared from plasma. Here, "plasma" means the supernatant obtained by adding centrifugation after adding anticoagulant, such as heparin and CPD, to the collected blood. The term "human serum prepared from plasma" refers to the preparation of plasma under centrifugal conditions (for example, 4,400 g x 5 minutes or more) in which the blood collected from the subject is completely precipitated by platelets, thereby removing the coagulation factor contained in the plasma. Human serum obtained by

(3) Human serum for cell culture obtained from a fluid containing a blood-derived liquid component and a platelet containing a blood coagulation factor, wherein the content of the cell proliferation factor is higher for cell culture than human serum prepared by neglect. Human serum.

According to the invention of (3), stem cell growth can be efficiently carried out by increasing the content of the cell proliferation factor of the human serum according to the present invention more than when prepared by standing. Here, "human serum prepared by standing" refers to human serum obtained by centrifugation after leaving blood collected from the subject at room temperature for about 1 hour in a plastic container.

(4) The human serum for cell culture according to any one of (1) to (3), wherein the cell proliferation factor comprises at least one of PDGF-BB or TGF-β1.

According to the invention of (4), it is possible to proliferate stem cells more efficiently by including at least one of PDGF-BB or TGF-β1 having a higher cell proliferation ability among the cell proliferation factors described above. In addition, PDGF-BB refers to any one of the three forms of PDGF (PDGF-AA, PDGF-BB, PDGF-CC).

(5) The human serum for cell culture according to any one of (1) to (4), wherein the cell proliferation factor is obtained by contacting the fluid with a glass workpiece.

According to the invention of (5), in order for platelets in the fluid to be activated to release the cell growth factor, it is necessary to contact the fluid with foreign matter. In the present invention, by using such foreign matter as a glass workpiece, it is possible to release the cell growth factor more efficiently than in the case of polyethylene pellets. Here, a "glass processed body" means glass powder, glass beads, etc. It is preferable to make the shape of a glass workpiece into a substantially spherical shape from the point of suppressing breakage (hemolysis) of red blood cells, and damage of the apparatus used when preparing the serum which concerns on this invention.

In addition, in order to quickly activate activated factors such as platelets and coagulation factors, it is preferable to form the surface of the glass workpiece 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. It is not limited to these.

(6) The human serum for cell culture according to (5), wherein the glass workpiece is glass beads.

According to the invention of (6), it is possible to further activate platelets in the fluid by making the glass workpiece into glass beads. Among them, porous glass beads are more preferable because of the large contact area with the fluid. Moreover, it is preferable that the surface area of glass beads is 0.1 mm <2> / ml-25 mm <2> / ml with respect to 1 ml of fluids.

(7) The human serum for cell culture according to any one of (1) to (6), which is prepared without being contacted with outside air.

According to the invention of (7), the human serum is prepared so that it does not come into contact with the outside air, thereby reducing the risk of the prepared serum being contaminated by bacteria or microorganisms. Thereby, high stability serum can be prepared, and further safety can be ensured. In addition, the human serum according to the invention of (7) is preferably prepared using an apparatus in a form not in contact with the outside air.

(8) The human serum for cell culture according to any one of (1) to (7), which is used in a regenerative medicine method.

According to the invention of (8), when used in a cell culture medium, when inoculating and culturing stem cells collected from a subject, the cells can be cultured more quickly. In addition, since the cells can be cultured with autologous serum, the side effects and the like are less likely to occur, which is excellent in terms of safety.

As described above, since the human serum for cell culture according to the present invention contains a large amount of cell growth factors, it is possible to culture stem cells more efficiently than conventional serum. In addition, when the human serum is used for regenerative medicine, it is possible to safely and reliably reproduce the tissue and function of the subject.

Hereinafter, the present invention will be described in more detail.

Human serum for cell culture according to the present invention, a cell for preparing a serum for a cell culture method comprising a serum preparation step of preparing a serum containing a cell growth factor and a culture step of culturing the cells in the presence of the prepared serum It is preferable to prepare by the serum preparation device for culture.

The serum preparation device has a blood storage section for storing a fluid containing a blood-derived liquid component including at least a coagulation factor and platelets, the blood storage section has a cell having a serum-generating function to generate serum suitable for the cell culture process It is preferable that it is a serum preparation device for culture. Further, the serum preparation device is more preferably a closed system, that is, a device capable of preparing serum without contacting the outside air. Here, the serum production function means a function of increasing the content of proliferation factor in the serum to increase the recovery ability of the serum by activating platelets in the fluid, and is given by a coagulation promoting solid having a specific gravity greater than that of the fluid.

Specifically, it is preferable to prepare by the serum preparation apparatus shown below, but it is not specifically limited to this apparatus.

<Serum preparation device>

An apparatus for producing human serum for cell culture according to the present invention includes (i) a blood storage unit for storing blood, (ii) a component receiving unit including human serum for cell culture separated from blood stored in the blood storage unit, and ( Iii) a connection part for connecting the blood storage part and the component accommodating part so as not to contact the outside air, wherein the blood storage part includes a blood coagulation accelerator which contacts the blood to promote coagulation of the blood, and the blood coagulation accelerator is It is characterized by producing human serum for cell culture suitable for cell culture without contact.

The blood reservoir comprises means for sorting human serum for cell culture from stored blood.

The serum preparation device 1 shown in FIG. 1 is composed of the blood storage unit 10 and the component receiving unit 20 as main elements. Among them, the blood reservoir 10 and the component accommodating portion 20 are main body portions formed in a bag shape by fusion of two sheets made of a plastic material, for example, soft polyvinyl chloride, at the outer edge portion 11a. (11) and the glass workpiece 12 inserted in the main body part 11 are comprised.

In the main body portion 11, the glass workpiece 12 serving as the coagulation accelerator is set in a state capable of flowing within the main body portion 11, and has a substantially spherical shape made of, for example, soda glass. It is. Moreover, it is preferable that the surface area of the glass workpiece 12 becomes a relation which is 0.1 mm <2> / ml or more. By setting it as such a range, activation of platelets and a coagulation factor in blood is accelerated | stimulated.

In addition, when the surface area of the glass processed body 12 with respect to the blood volume which can be stored is made into 0.1 mm <2> / ml or more and 25.0 mm <2> / ml, suppression of hemolysis in an activation acceleration | stimulation process and a centrifugation process, and platelet and a coagulation factor It becomes possible to be compatible with the promotion of activation.

More preferably, a layer of a silicon dioxide compound composed of at least one selected from glass, silica, silicon earth and kaolin is formed on the surface of the glass beads, and the glass beads are spherical or substantially spherical. It is good to be.

In addition, two tubes 41 and 42 are hermetically connected to an upper end of the main body portion 11 of the blood reservoir 10 at its connection port. Among them, the tube 41 serves as an introduction for introducing blood, so that the other end is connected to a blood collection needle 30 or a connection portion that can be connected to the blood collection needle. Such a structure makes it possible to prepare the collected blood with serum without contacting the outside air.

Another tube 42, which is hermetically connected to the blood storage unit 10, is a self-supporting body 21 through tubes 43 to 46, 51 to 56, and branch bodies 61 to 65. To (26). They serve as derivation paths for deriving separated blood components. These tubes 41 to 46 and 51 to 56 are made of a flexible resin material such as soft polyvinyl chloride. Here, the bag-shaped bodies 21 to 26 and the tubes 51 to 56 of the component accommodating portion 20 are also hermetically contacted by solvent bonding, heat fusion, or ultrasonic welding.

<Serum preparation process>

The method for producing human serum for cell culture according to the present invention comprises the steps of: (i) storing blood containing blood coagulation factors and platelets in the blood storage unit, (ii) adding a blood coagulation promoter in the blood storage unit in which the blood is stored. After the activation and promotion step of stirring and contacting the blood and the blood coagulation promoting agent and (iii) the separation and storage process of separating and extracting human serum for cell culture from the blood undergoing the activation promotion process and kept in contact with the outside air It is characterized by.

A serum preparation process using the blood preparation device 1 having the above-described configuration will be described with reference to FIGS. 2 and 3.

As shown in Fig. 2, the blood separation operation using the serum preparation device 1 is roughly divided into seven steps (S1 to S7).

First, in the first step of the operation, the blood needle 30 in FIG. 1 is stabbed by the subject (patient) to collect blood. At this time, the blood collected from the blood collection needle 30 is stored in the blood storage unit 10 located below through the tube 41 (storage step S1). Here, the path is closed at the source side of the blood storage unit 10 by using a clamp or the like so that blood collected in the blood storage unit 10 does not flow into the component receiving unit 20. The storage step (S1), after taking the required amount in consideration of the physical condition of the patient at the time of blood collection and ends. The requirement here refers to about 200 ml to 600 ml when there is no problem in the size or physical condition of the patient.

Next, as shown in Fig. 2, after starting the storage step S1, the blood storage unit 10 is shaken in parallel with it (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 workpiece 12 housed therein. Then, platelets and coagulation factors contained in the blood coagulate on the surface of the glass workpiece 12, and proliferation factors derived therefrom are released from the platelets activated during coagulation (in addition, the activation promoting process is carried out at low temperature. Is effective in promoting platelet aggregation). After the storage step S1, the blood collection needle 30 is removed from the blood collection subject, and a portion of the tube 41 connecting the blood collection needle 30 and the blood storage unit 10 is melted and at the same time the tip of the blood extraction is removed. Welding is carried out (melting step S3).

On the other hand, the blood storage unit 10 separated from the subject is an activation-promoting process together with the component receiving unit 20 and each of the tubes 42 to 46, 51 to 56, and the branching bodies 61 to 65 connecting them. Collected compactly through (2) and placed in a centrifuge (centrifugation step S4). The conditions of the centrifugal separation of the blood storage unit 10 are set according to the amount of stored blood and the kind of the component to be separated, but are set at, for example, 2,250 g × 10 minutes and 4 ° C. In addition, at this time, the tube 42 is preserve | saved in the state in which the path | route was closed by the partition or clamp which can be broken similarly to the case of the storage process S1.

Melting step (S3) and centrifugation step (S4) may be carried out before the activation promoting step (S2) when the anticoagulant is added in advance and the blood is collected. In this case, centrifugation is preferably performed under the following conditions.

Centrifugation of whole blood: 4,400 g × 4 to 6 minutes, 2,250 g × 10 minutes

Centrifugation of multiplatelet plasma (PRP): 1,100 g × 4 to 6 minutes

Returning to FIG. 2, the activated factor including the blood cell component activated in the centrifugation step S4 from the activation promotion step 2 is agglomerated and separated from the blood (separation step S5). In addition, in the separating step (S5), the serum 71 separated and extracted in the blood storage unit 10 is pressed against the blood storage unit 10 to all or part of each bag 21 to 26. Are subdivided sequentially (derivation step S6).

As shown in FIG. 2, after the required amount of serum is filled in the bag 21, the tube 51 is melted and welded (melting step S7). This melting and welding are performed using the same method as that which melted and welded the tube 42 before the centrifugation process S4. In addition, the bag 21 filled with serum is subjected to a preservation treatment such as cryopreservation.

This derivation step (S6) and the melting step (S7) are carried out sequentially for each of the bag-shaped bodies 21 to 26, and at the time when all or part of the bag-shaped bodies 21 to 26 are filled with serum, End the operation. In addition, red blood cells may be washed and diluted with blood preservatives such as anticoagulants such as physiological saline, CPD solution, ACD-A solution, MAP solution, or the like, and stored as hemostatic blood, if necessary.

Example

Hereinafter, the present invention will be described in more detail, but the present invention is not limited to these examples.

<Example 1>

20 ml of fresh human blood was put into the container which put five glass beads ((phi): 4mm, 50mm <2>), and it shaken by the multi-shaker (MMS-300 Tokyo cooking equipment). 10, 20, 30 and 60 minutes after the start of shaking, 1 ml of blood was sampled into a sampling tube containing anticoagulant and serum was separated by centrifugation. In addition, CPD-added blood was collected from the same subject, and plasma was separated. Calcium chloride was added to the separated plasma and fibrin was precipitated at 37 ° C to prepare serum. Proliferation factors (PDGF-BB, TGF-β1) were measured for serum prepared from containers containing glass beads and serum prepared from plasma isolated from CPD-added blood. The results are shown in Table 1. In addition, the growth factor was measured using a test kit manufactured by R & D SYSTEMS and a microplate reader (manufactured by Multiscan BICHROMATIC Labsystem).

Difference in Proliferation Factor Contents According to Serum Derivatives  Origin of Serum  Derived from a container containing five glass beads   CPD-added plasma derived  Shaking time  10 minutes  20 minutes  30 minutes  60 minutes  PDGF-BB  40.5 pg / ml  753.7 pg / ml  1677.2 pg / ml  1677.2 pg / ml  <32.5 pg / ml  TGF-β1  21.2 ng / ml  24.4 ng / ml  26.4 ng / ml  29.6 ng / ml  1.5 ng / ml

For serum prepared in a container of five glass beads, the PDGF-BB content rose with the passage of shaking time, and after 60 minutes 1677.2 pg per ml of serum was released. At this time, the amount of PDGF-BB contained in the plasma derived serum of the same subject was less than the detection limit of 32.5 pg / ml. TGF-β1 released 21.2 ng of TGF-β1 per 1 ml of serum at 10 minutes from the start of shaking in the case of serum prepared in a container containing 5 glass beads, and then gradually increased gradually over time, 60 After 2 minutes it reached 29.6 ng / ml. On the other hand, the amount of TGF-β1 released from plasma of the same subject was 1.5 ng per ml of serum.

<Example 2>

Three vessels were prepared: a blood collection container (sample 3) not containing glass beads, a blood collection container (sample 1) containing five glass beads, and a blood collection container (sample 2) containing 20 polyethylene pellets. 20 mL of fresh blood from the same subject was put in each container, and it shaken by the multi-shaker (MMS-300, Tokyo cooking equipment). After 10, 20, 30, and 60 minutes of shaking, 1 ml of blood was sampled into a sampling tube containing anticoagulant, and a platelet was counted using a blood cell counting device (manufactured by multi-item automatic blood counting device K-4500 Sysmax). The number was measured.

In addition, each sample was centrifuged to separate serum, and growth factors (PDGF-BB and TGF-β1) in serum were measured. The growth factor was measured using a test kit manufactured by R & D SYSTEMS and a microplate reader (manufactured by Multiskan BICHROMATIC Labsystem).

In addition, blood was collected from a commercially available vacuum test tube (manufactured by Benozect II Termosa) from the same subject, and serum was separated after standing for 1 hour at room temperature, and the growth factor was similarly quantified. For platelet count, the value when 100% of the value immediately after blood collection was expressed as platelet residual rate. For each growth factor, the amount of growth factor in serum prepared by the vacuum collection vessel is defined as the potential amount of growth factor in platelets, and the amount of growth factor in serum obtained at each shaking time is expressed as the ratio of the potential amount as the release rate. It is shown.

[Platelet count]

Fig. 4 is a diagram showing the shaking time and the platelet survival rate in each sample. Within 20 minutes after the onset of shaking, the platelet survival rate in Sample 1 decreased to about 2%. On the other hand, in the case of sample 2, it decreased within 30 minutes after the start of shaking slightly later than sample 1. In addition, in the case of sample 3, the platelet survival rate decreased sharply immediately after the onset of shaking, but only 60% after the onset of shaking.

[Release Rate of PDGF-BB]

Fig. 5 is a graph showing the relationship between the shaking time and the release rate of PDGF-BB in each sample. Sample 1 rapidly released PDGF-BB over time after the onset of shaking, and about 90% of the serum potential was released after shaking for 1 hour. For the other two samples, despite the decrease in platelet count, the release of PDGF-BB did not release 20% of the potential in serum.

[Release Rate of TGF-β1]

Fig. 6 shows the relationship between the shaking time and the release rate of TGF-β1 in each sample. In the serum of Sample 1, TGF-β1 was released in the amount of 70% of the latent amount in serum from 10 minutes after the start of shaking. In the other two samples, it was confirmed that the release amount tended to increase, but even after 60 minutes, the release rate remained at about 40% of the latent amount in the serum. From the above results, polyethylene pellets also play the same role as glass beads in removing platelets from blood cells, but they cannot activate platelets.

1 is a diagram showing a serum preparation device for preparing human serum for cell culture according to the present invention.

2 is a view showing a procedure for preparing human serum for cell culture according to the present invention.

3 is a diagram for shaking the blood storage unit 10 of the serum preparation device.

Fig. 4 is a diagram showing the shaking time and the platelet survival rate in each sample.

5 is a graph showing the relationship between the shaking time and the release rate of PDGF-BB in each sample.

Fig. 6 is a graph showing the relationship between the shaking time and the release rate of TGF-β1 in each sample.

Claims (8)

(Iii) a blood reservoir for storing blood, (Ii) a component receptacle comprising human serum for cell culture isolated from blood stored in said blood reservoir; (Iii) a connection part for connecting the blood storage part and the component accommodating part so as not to contact the outside air, wherein the blood storage part includes a blood coagulation accelerator for contacting blood to promote coagulation of blood, The blood coagulation promoter produces a human serum for cell culture, characterized in that for producing a cell culture suitable for cell culture without contact with outside air. The apparatus of claim 1, wherein the blood storage unit comprises a means for classifying the human serum for cell culture from the stored blood. The apparatus of claim 1, wherein the blood coagulation promoter is glass beads having a surface area of 0.1 mm 2 to 25 mm 2 with respect to 1 ml of blood. The cell culture human according to claim 3, wherein a layer of a silicon dioxide compound composed of at least one selected from glass, silica, silicon earth and kaolin is formed on the surface of the glass beads. Serum production apparatus. The apparatus for producing human serum for cell culture according to claim 3 or 4, wherein the glass beads are spherical. (Iii) storing blood, including coagulation factors and platelets, in the blood reservoir; (Ii) an activation promoting step of injecting a blood coagulation accelerator into the blood storage unit where blood is stored and then stirring and contacting the blood and the coagulation accelerator; (Iii) separating and extracting the human serum for cell culture from the blood that has undergone the activation promotion process, and then separating and storing the serum to prevent contact with the outside air. The method of claim 6, wherein the coagulation promoter is glass beads having a surface area of 0.1 mm 2 to 25 mm 2 with respect to 1 ml of blood. The method for producing human serum for cell culture according to claim 6 or 7, wherein the human serum for cell culture is separated and extracted from the blood which has undergone the activation promotion step by centrifugal separation.

Images