JP7025070B1 - Blood-derived growth factor-containing composition and its preparation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a blood-derived growth factor-containing composition and a method for preparing the blood-derived growth factor-containing composition, which are more effective for wound healing and tissue regeneration.
One embodiment of the present invention is a method of preparing a blood-derived growth factor-containing composition from mammalian blood.
The method includes a step of adding an anticoagulant capable of forming a chelate complex with a metal ion to the blood, and a step of separating platelet-rich plasma containing a buffy coat component from the blood to which the anticoagulant is added. It comprises a step of freezing and thawing the platelet-rich plasma to activate it.
[Selection diagram] None

Description

The present invention relates to a blood-derived growth factor-containing composition and a method for preparing the same.

There is a treatment method using platelet-rich plasma (hereinafter, also referred to as "PRP"), which is plasma enriched with platelets prepared by centrifuging its own blood. This platelet-rich plasma is rich in various growth factors. Platelet-rich plasma is a promising material in the field of regenerative medicine because these growth factors play an effective role in wound healing and tissue regeneration (for example, Patent Document 1).

The preparation of PRP includes a step of activating platelets and releasing growth factors and the like contained in the platelets. Platelet-derived factor release stimulants such as thrombin and calcium chloride are used for platelet activation.

Japanese Unexamined Patent Publication No. 2009-195739

A main object of the present invention is to provide a blood-derived growth factor-containing composition and a method for preparing the blood-derived growth factor-containing composition, which are effective for wound healing and tissue regeneration. Further, the present invention has a sub-problem to provide a blood-derived growth factor-containing composition capable of relieving strong pain during administration and a method for preparing the blood-derived growth factor-containing composition.

As a result of diligent research on the above-mentioned main subject, it is important for the present inventors to pay attention to a specific growth factor {vascular endothelial growth factor (hereinafter, also referred to as "VEGF")} and its amount. I found that there was. Furthermore, the present inventors can select a specific raw material and process (use of chelating agent, collection of buffy coat layer, freeze-thaw) from a myriad of combinations of raw materials and processes to obtain the specific growth factor. It has been found that it is possible to prepare a blood-derived growth factor-containing composition containing a desired amount or more. Furthermore, as a result of diligent research on the above-mentioned sub-problems, the present inventors have obtained a blood-derived growth factor-containing composition capable of alleviating strong pain during administration by also performing filtration treatment with a filter of a predetermined size. It has been found that it can be prepared. Specifically, it is the following invention.

That is, according to the present invention.
[1] A blood-derived growth factor-containing composition prepared from mammalian blood and having a growth factor VEGF content of 100 pg / mL or more;
[2] Anti-inflammatory cytokine: Interleukin-1 receptor antagonist (hereinafter, also referred to as "IL-1Ra") content is 100 pg / mL or more, derived from the blood according to the above [1]. Growth factor-containing composition;
[3] Inflammatory cytokines: interleukin-1β (Interleukin-1β: hereinafter also referred to as "IL-1β"), interleukin-6 (Interleukin-6: hereinafter also referred to as "IL-6"), and tumor necrosis factor. The content of the factor (Tumor necrosis factor-α: hereinafter also referred to as “TNF-α”) is 10 pg / mL or less, 10 pg / mL or less, and 20 pg / mL or less, respectively, [1] or [2]. The blood-derived growth factor-containing composition described;
[4] The blood-derived growth factor according to any one of [1] to [3] above, wherein the mass ratio of the anti-inflammatory cytokine IL-1Ra based on the mass of the growth factor VEGF is 1: 1 to 50. Composition;
[5] The item according to any one of [1] to [4] above, wherein the mass ratio of the inflammatory cytokine IL-1β based on the mass of the growth factor VEGF is 1: 0.001 to 0.05. Blood-derived growth factor composition;
[6] The blood-derived growth factor composition according to any one of [1] to [5] above, which is in a freeze-dried state;
[7] A method for preparing a blood-derived growth factor composition from mammalian blood, wherein an anticoagulant capable of forming a chelate complex with a metal ion is added to the blood, and the anticoagulant is added. A method comprising a step of separating multiplatelet plasma containing a buffy coat component from the blood and a step of freeze-thawing and activating the polyplatelet plasma;
[8] The method according to [7] above, wherein the anticoagulant contains citric acid, EDTA, or a salt thereof.
[9] In the step of separating platelet-rich plasma from the blood, the blood is subjected to the first centrifugation treatment at 100 to 1000 G for 1 to 30 minutes to recover the upper layer including the plasma fraction and the buffy coat. And, the upper layer is subjected to a second centrifugation treatment of 1000 to 2500 G for 1 to 30 minutes to pelletize the plasma and the buffy coat component; the supernatant is removed and the plasma and the buffy coat component are suspended. The method according to [7] or [8] above, which comprises turbidity to obtain the platelet-rich plasma;
[10] The method according to [9] above, wherein in the step of recovering the upper layer portion, the volume ratio to be recovered is 1: 0.1 to 0.5 with respect to the volume of the total liquid volume.
[11] The above-mentioned [9] or [10], wherein in the step of removing the supernatant, the supernatant having a volume ratio of 1: 1 to 10 is left with reference to the volume of the pellet, and the rest is removed. the method of;
[12] The step of freeze-thawing and activating the platelet-rich plasma is: freezing by treatment at −200 ° C. to −20 ° C. for 10 minutes or longer, and then thawing by treatment at 20 ° C. to 50 ° C. for 10 minutes or longer. The method according to any one of the above [7] to [11], which is carried out by the above;
[13] In any of the above [7] to [12], further comprising a step of pre-refrigerating the blood to which the anticoagulant is added at 0 to 10 ° C. before the step of separating the platelet-rich plasma. Method of description;
[14] The above-mentioned [7] to [13], further comprising a step of filtering the activated platelet-rich plasma using a filter having a pore size of 0.2 to 1 μm. The method described in either;
[15] The method according to any one of [7] to [14] above, further comprising a step of freeze-drying the activated platelet-rich plasma is provided.

According to the present invention, it is possible to provide a blood-derived growth factor-containing composition and a method for preparing the blood-derived growth factor-containing composition, which are effective for wound healing and tissue regeneration. Furthermore, according to the present invention, it is possible to provide a blood-derived growth factor-containing composition capable of relieving strong pain during administration and a method for preparing the blood-derived growth factor-containing composition.

FIG. 1 is a graph showing the amounts of growth factors and anti-inflammatory cytokines in the blood-derived growth factor-containing compositions of Examples 6-10.

Hereinafter, the present invention will be described in detail. Hereinafter, a method for preparing a blood-derived growth factor-containing composition according to the present embodiment will be described, and then a blood-derived growth factor-containing composition according to the present embodiment obtained by the above preparation method will be described.

<< Preparation method of blood-derived growth factor-containing composition >>
The method for preparing a blood-derived growth factor-containing composition according to the present embodiment is a method for preparing a blood-derived growth factor composition from mammalian blood, wherein an anticoagulant capable of forming a chelate complex with a metal ion is used in the blood. A step of adding to the blood (hereinafter, also referred to as "addition step") and a step of separating multi-platelet plasma containing a buffy coat component from the blood to which the anticoagulant is added (hereinafter, also referred to as "separation step"). , A method including at least a step of freeze-thawing and activating the polyplatelet plasma (hereinafter, also referred to as a “freeze-thaw step”). Here, the "mammalian blood" is not particularly limited as long as it contains a growth factor, and is preferably derived from humans. In addition, the "mammalian blood" is preferably self-derived. Hereinafter, each step described above will be described in detail.

<Addition process>
In the method of this embodiment, blood to which an anticoagulant that forms a chelate complex with a metal ion is added is used. The anticoagulant may be added to the collected blood later, but it is desirable to collect the blood in advance in a container such as a blood collection tube or a blood collection bag containing the anticoagulant.

Examples of the anticoagulant that forms a chelate complex with a metal ion include a compound that forms a chelate complex with a metal ion (for example, calcium ion), for example, a polycarboxylic acid and a salt thereof. Examples of the polycarboxylic acid include citric acid and EDTA. Examples of the polycarboxylic acid salt include EDTA salts such as 2Na citrate (citrate), EDTA-2K, and EDTA-2Na.

The anticoagulant may be added as a solid, or may be added as a solution in which the anticoagulant is dissolved in a solvent such as pure water (preferably one that is physiologically acceptable). The final concentration of the anticoagulant in the whole blood after the addition of the anticoagulant (whole blood + anticoagulant or whole blood + anticoagulant solution) is preferably 0.5 in the case of 2Na citrate. It is ~ 1.5% by mass, more preferably 0.8 to 1.0% by mass, and in the case of EDTA-2K, it is preferably 0.5 to 2.0 mg / mL, still more preferably 1.1 to 1. It is .7 mg / mL, and in the case of EDTA-2Na, it is preferably 0.5 to 2.0 mg / mL, and more preferably 1.0 to 1.5 mg / mL.

<Pre-refrigerating process>
The method of the present embodiment further includes a step of pre-cooling the blood to which the anticoagulant is added before the step of separating the polyplatelet plasma from the blood to which the anticoagulant forming a chelate complex with the metal ion is added. It may be included. The refrigerating temperature in the step of pre-refrigerating blood is, for example, 0 to 10 ° C. The process time of the step of pre-refrigerating blood is, for example, more than 0 hours and 72 hours or less. The lower limit of the process time may be 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, and 8 hours or more. The upper limit of the process time may be 60 hours or less, 48 hours or less, 36 hours or less, and 24 hours or less.

<Separation process>
The method of the present embodiment includes the step of separating the platelet-rich plasma containing the buffy coat component from the blood to which the anticoagulant is added. Here, the "buffy coat" refers to a thin white layer containing a large amount of leukocytes formed between erythrocytes and plasma fractions when blood is centrifuged. Further, "containing a buffy coat component" means containing at least a part of the buffy coat.

During this separation step, the temperature of blood and platelet-rich plasma is preferably controlled at 4-25 ° C.

The means for separating platelet-rich plasma from blood (whole blood) is not limited, and any known means can be used. Usually, the blood is subjected to a relatively slow first centrifugation treatment to separate the erythrocyte-containing fraction, the buffy coat, the platelet-rich plasma (PRP), and the platelet-rich plasma (platelet power plasma). Then, platelet-rich plasma is obtained. Here, it is preferable that the separation step includes a relatively low-speed first centrifugation treatment and a relatively high-speed second centrifugation treatment. The first centrifugation treatment and the second centrifugation treatment may be independently performed a plurality of times. Alternatively, platelet-rich plasma and buffy coat may be obtained by the first centrifugation treatment at a relatively low rate. Hereinafter, the first centrifugation treatment, the second centrifugation treatment, and the concentration / purification treatment of platelets and the buffy coat component will be described in detail.

(First centrifugation treatment)
The first centrifuge treatment at a relatively low speed is a centrifuge of 100 to 1000 G (corresponding to about 200 to 2000 rpm in a normal centrifuge). The rotation conditions may be 150 to 750 G and 200 to 500 G. The processing time may be 1 to 30 minutes, 5 to 20 minutes, and 10 to 15 minutes.

After the first centrifugation treatment at a relatively low rate, the entire plasma fraction (platelet rich plasma and platelet rich plasma) and the upper layer containing the buffy coat are collected.

In the step of recovering the upper layer portion, an arbitrary volume amount can be recovered so as not to contain a fraction containing red blood cells as much as possible (for example, visually). Specifically, for example, the volume ratio of the upper layer to be recovered is preferably 1: 0.1 to 0.5, and more preferably 1: 0.2 to 0. It is 5, and more preferably 1: 0.3 to 0.5.

(Second centrifuge treatment)
Platelets and buffy coat components are pelleted by a relatively high speed second centrifugation treatment on the upper layer recovered after the first centrifugation treatment.

The second centrifuge treatment at a relatively high speed is a centrifuge of 1000 to 2500 G (corresponding to about 2000 to 5000 rpm in a normal centrifuge). The rotation conditions may be 1100 to 2000 G, 1200 to 1500 G. The processing time may be 1 to 30 minutes, 5 to 20 minutes, and 10 to 15 minutes.

(Platelet and buffy coat component purification / concentration treatment)
Platelets and buffy coat components can be purified and concentrated by removing the supernatant from the one obtained by the second centrifugation treatment and then suspending the supernatant. Here, in the step of removing the supernatant, any volume can be removed. Specifically, for example, the volume ratio is preferably 1: 1 to 10, more preferably 1: 1 to 5, and even more preferably 1: 1 to 3 based on the volume of the pellet. The supernatant can be left and the rest can be removed.

<Freezing and thawing process>
This step activates platelet-rich plasma and releases numerous growth factors and anti-inflammatory cytokines.

Addition of a drug is known as a method for activating platelet-rich plasma. Examples of the drug that activates platelet-rich plasma include calcium ion source compounds such as calcium chloride, calcium phosphate, calcium lactate, and calcium carbonate. In the method of the present embodiment, activation of platelet-rich plasma can be achieved by freezing and thawing without adding a drug. Therefore, the use of calcium ion source compounds can be reduced as much as possible and the amount of additives can be minimized.

Freezing of platelet-rich plasma can be performed by exposing the platelet-rich plasma to a low temperature at which freezing occurs. Specifically, by placing the platelet-rich plasma under temperature conditions of, for example, −10 ° C. or lower, −20 ° C. or lower, −100 ° C. or lower, or −200 ° C. or lower, preferably −20 ° C. to −200 ° C. Can be frozen. This may be performed by contact with liquid nitrogen or storage in a freezer.

The time of the step of freezing the platelet-rich plasma and activating the platelets is not particularly limited. The freezing process time may be 1 minute or longer, 5 minutes or longer, 10 minutes or longer, 15 minutes or longer, 20 minutes or longer, 25 minutes or longer, or 30 minutes or longer. On the other hand, it may be 180 minutes or less, 150 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, and 30 minutes or less. Specifically, for example, freezing can be carried out by immersing a container containing platelet-rich plasma in liquid nitrogen for 1 minute or longer.

Melting of platelet-rich plasma can be performed by exposing the platelet-rich plasma to a temperature at which thawing occurs. Specifically, the temperature of frozen platelet-rich plasma is, for example, 20 ° C. or higher, 25 ° C. or higher, or 30 ° C. or higher, and 50 ° C. or lower, 40 ° C. or lower, or 38 ° C. or lower, preferably 20 ° C. to 50 ° C. Melting can be performed by placing it under conditions. The time of the step of thawing platelet-rich plasma and activating platelets is not particularly limited. The lower limit of the melting process time may be 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, and 30 minutes or more. On the other hand, the upper limit of the melting process time may be 180 minutes or less, 150 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, and 30 minutes or less. Specifically, for example, platelets can be activated by allowing a container containing platelet-rich plasma to stand at room temperature for 10 to 30 minutes.

A solvent may be added to the activated platelet-rich plasma to resuspend it. The solvent to be resuspended is arbitrary as long as it is physiologically acceptable. For example, plasma, physiological saline, buffered physiological saline {for example, phosphate buffered saline (PBS)}, Ringer's solution (for example, Lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution) and the like can be mentioned. In addition, any platelet resuspension resuspended in any solvent is referred to as "platelet rich plasma" in the present specification. Further, the above solvent may be further added to this resuspension for the purpose of adjusting the concentration or blending additives, and the liquid after the addition is also referred to as "platelet rich plasma".

<Cell removal process>
The method of this embodiment may further include the step of removing blood cells from platelet-rich plasma.

During this cell removal step, the platelet-rich plasma is preferably controlled at 4-25 ° C.

Platelet-rich plasma contains platelets. Further, according to the method of the present embodiment, the platelet-rich plasma also contains leukocytes derived from a buffy coat. In addition, the platelet-rich plasma may contain a small amount of red blood cells.

Means for removing blood cells include, for example, heat treatment, acid treatment, filtering treatment and the like. Of these, filtering treatment is preferable because the risk of causing denaturation of growth factors is small. That is, this step is preferably a filtration treatment step of removing blood cells from platelet-rich plasma by a filtering treatment.

The filter used for the filtering process is not limited as long as it can remove blood cells. Since the size of blood cells is the smallest when platelets are about 2 μm, for example, a membrane filter having a pore size of 1 μm or less, 0.7 μm or less, and 0.5 μm or less can be used. There is no limit to the lower limit of the pore size of the membrane filter, but if it is excessively small, the risk of clogging increases. Therefore, the pore diameter is preferably 0.2 μm or more, 0.3 μm or more, and 0.4 μm or more. Among the commercially available membrane filters, those satisfying this range include a membrane filter having a pore diameter of about 0.2 μm and a membrane filter having a pore diameter of about 0.45 μm. Any of these can be used in this embodiment, and a membrane filter having a pore size of about 0.45 μm is the optimum membrane filter.

The filtering process may be performed by centrifugation using a centrifuge tube provided with the above-mentioned filter. The centrifuge at this time is, for example, a centrifuge of 800 to 1800 G (corresponding to about 1500 to 4000 rpm in a normal centrifuge). The process time is, for example, 1 to 30 minutes, 5 to 15 minutes, and 10 minutes.

As a result of this treatment, platelet-rich plasma that has passed through the filter and is substantially free of blood cells can be obtained.

<Freeze-drying process>
The method of this embodiment may further include a step of freeze-drying platelet-rich plasma.

The freeze-drying method is not particularly limited as long as it is a conventionally known protein freeze-drying method.

For freeze-drying, platelet-rich plasma is freeze-dried and then vacuum freeze-dried.

In the freezing process, the temperature or environmental temperature of the platelet-rich plasma is adjusted to be −60 ° C. or lower, −70 ° C. or lower, or −80 ° C. or lower. This may be performed by contact with liquid nitrogen or storage in a freezer.

The execution time of the freezing process may be instantaneous. Usually, it is carried out for 6 to 24 hours at the above temperature of the freezing process to ensure that all platelet-rich plasma is frozen. That is, the interval between the start time of the freezing process and the start time of the vacuum freeze-drying process may be 0 to 24 hours or 6 to 24 hours.

The temperature conditions for the vacuum freeze-drying treatment are not particularly limited. The upper limit of the temperature condition may be −30 ° C. or lower, −35 ° C. or lower, −40 ° C. or lower, −45 ° C. or lower. The lower limit is not particularly limited, but may be −80 ° C. or higher, −75 ° C. or higher, −70 ° C. or higher, −65 ° C. or higher, −55 ° C. or higher, −50 ° C. or higher.

The atmospheric pressure conditions for the vacuum freeze-drying treatment are not particularly limited. The upper limit of the atmospheric pressure condition may be 20 Pa or less, 18 Pa or less, 16 Pa or less, and 15 Pa or less. The lower limit value is 0 Pa or more, and may be 1 Pa or more, 2 Pa or more, 3 Pa or more, 4 Pa or more, and 5 Pa or more.

The time for executing the vacuum freeze-drying treatment is not particularly limited, and the time can be set according to the amount of liquid. The lower limit of the freeze-drying treatment time may be, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, and 8 hours or more. On the other hand, the upper limit may be 24 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, and 8 hours or less.

The means for performing the vacuum freeze-drying treatment is not particularly limited, and any means can be used. For example, FDU-1110 (manufactured by Tokyo Rika Kikai Co., Ltd.) may be used for execution.

After the vacuum freeze-drying treatment, almost completely dried granular or powdered platelet-rich plasma can be obtained.

≪Blood-derived growth factor-containing composition≫
The blood-derived growth factor-containing composition according to the present embodiment is a blood-derived growth factor-containing composition prepared from mammalian blood, and is a blood-derived growth factor-containing composition having an extremely high content of a specific growth factor. be. The blood-derived growth factor-containing composition may be a liquid, a semi-solid, or a dry solid. Hereinafter, the contained components will be described in detail.

<Growth factor>
(VEGF)
The blood-derived growth factor-containing composition according to the present embodiment has a growth factor VEGF content of at least 90 pg / mL, preferably 100 pg / mL or more, more preferably 200 pg / mL or more, still more preferably 200 pg / mL or more. It is 300 pg / mL or more. The upper limit varies depending on the individual difference of the mammal from which the blood was collected, but is, for example, 10,000 pg / mL. Here, "pg / mL" in the present specification and the scope of the present patent is the concentration in the platelet-rich plasma after the filtration treatment step, or 300 mg of the platelet-rich plasma after the freeze-drying step is thawed per 1 mL of the solvent. Concentration in platelet-rich plasma. VEGF is a growth factor that is also contained in platelets but is abundant in buffy coats. VEGF has an angiogenic effect, and a blood-derived growth factor-containing composition containing a large amount of VEGF can be expected to have a higher wound healing effect. The VEGF content (pg / dried product 1 g) with respect to the dry mass of the composition is preferably 300 pg / g or more, more preferably 700 pg / g or more, and further preferably 1000 pg / g or more. be. The upper limit varies depending on the individual difference of the mammal from which the blood was collected, but is, for example, 30,000 pg / g. Here, when the composition is not in a dry form, the dry mass of the composition is the mass after the composition is dried under the following drying conditions.
(Drying conditions)
Using FDU-1110 (manufactured by Tokyo Rika Kikai Co., Ltd.), the composition to be dried is subjected to vacuum freeze-drying treatment under the conditions of 5 Pa and −45 ° C. for 16 hours.

(Other growth factors)
The blood-derived growth factor-containing composition according to the present embodiment may contain other growth factors. The type of growth factor is not particularly limited, but is, for example, platelet-derived growth factor (eg PDGF-AB and PDGF-BB), epithelial growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (eg, eg). TGF-β), insulin-like growth factor (IGF), hepatocellular growth factor (HGF).

<Anti-inflammatory cytokine>
(1L-1Ra)
The blood-derived growth factor-containing composition according to this embodiment may contain the anti-inflammatory cytokine IL-1Ra. The content of IL-1Ra is preferably 100 pg / mL or more, more preferably 150 pg / mL or more, and further preferably 200 pg / mL or more. The upper limit varies depending on the individual difference of the mammal from which the blood was collected, but is, for example, 50,000 pg / mL. IL-1Ra is mainly contained in leukocytes. IL-1Ra acts as an antagonist of the inflammatory cytokines IL-1α and IL-1β and suppresses inflammation. The content (pg / 1 g of dried product) of the anti-inflammatory cytokine IL-1Ra with respect to the dry mass of the composition is preferably 300 pg / g or more, more preferably 450 pg / g or more, still more preferable. Is 600 pg / g or more. The upper limit varies depending on the individual difference of the mammal from which the blood was collected, but is, for example, 150,000 pg / g. Here, when the composition is not in a dry form, the dry mass of the composition is the mass after the composition is dried under the above-mentioned drying conditions.

<Inflammatory cytokine>
The blood-derived growth factor-containing composition according to this embodiment may contain the inflammatory cytokines IL-1β, IL-6 and TNF-α. The contents of IL-1β and IL-6 are independently, preferably 10 pg / mL or less, more preferably 9.5 pg / mL or less, and further preferably 9.0 pg / mL or less. The content of TNF-α is preferably 20 pg / mL or less, more preferably 19 pg / mL or less, still more preferably 18 pg / mL or less. Overexpression of these inflammatory cytokines can lead to the development of diseases such as rheumatoid arthritis. The inflammatory cytokine contained in the blood-derived growth factor-containing composition according to the present embodiment is less likely to cause such side effects even when administered to the human body by setting the concentration to a low concentration as described above.
The contents of IL-1β and IL-6 (pg / 1 g of dried product) with respect to the dry mass of the composition are independently, preferably 30 pg / g or less, and more preferably 28 pg / g or less. Yes, more preferably 27 pg / g or less. Further, the content of TNF-α (pg / 1 g of dried product) with respect to the dry mass of the composition is preferably 60 pg / g or less, more preferably 57 pg / g or less, still more preferably 54 pg / g. It is as follows. Here, when the composition is not in a dry form, the dry mass of the composition is the mass after the composition is dried under the above-mentioned drying conditions.

<Component ratio>
(Growth factors and anti-inflammatory cytokines)
The blood-derived growth factor-containing composition according to the present embodiment preferably contains growth factors and anti-inflammatory cytokines in a well-balanced manner. Specifically, the mass ratio of the anti-inflammatory cytokine IL-1Ra based on the mass of the growth factor VEGF is preferably 1: 1 to 50, more preferably 1: 1 to 15, and even more preferably. It is 1: 1 to 10. A blood-derived growth factor-containing composition containing a growth factor and an anti-inflammatory cytokine in such a ratio has both a wound healing effect and an anti-inflammatory effect, and therefore exhibits a higher therapeutic effect when administered to the human body.

(Growth factors and inflammatory cytokines)
The blood-derived growth factor-containing composition according to the present embodiment preferably contains a large amount of growth factors and hardly contains inflammatory cytokines. Specifically, the mass ratio of the inflammatory cytokine IL-1β based on the mass of the growth factor VEGF is preferably 1: 0.001 to 0.05, more preferably 1: 0.001 to 0. It is 03, and more preferably 1: 0.001 to 0.01. A blood-derived growth factor-containing composition containing a growth factor and an inflammatory cytokine in such a ratio is less likely to cause inflammation when administered to the human body, and has a high therapeutic effect.

≪Freeze-dried product≫
The blood-derived growth factor-containing composition according to this embodiment may be in a freeze-dried state.

<Growth factor>
(VEGF)
The blood-derived growth factor-containing composition in the freeze-dried state according to the present embodiment has a growth factor VEGF content of preferably 300 pg / g or more, more preferably 700 pg / g or more, still more preferably 1000 pg / g. That is all. The upper limit varies depending on the individual difference of the mammal from which the blood was collected, but is, for example, 30,000 pg / g.

(Other growth factors)
The blood-derived growth factor-containing composition in the lyophilized state according to the present embodiment may contain other growth factors. The type of growth factor is not particularly limited, but is, for example, platelet-derived growth factor (eg PDGF-AB and PDGF-BB), epithelial growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (eg, eg). TGF-β), insulin-like growth factor (IGF), hepatocellular growth factor (HGF).

<Anti-inflammatory cytokine>
(1L-1Ra)
The blood-derived growth factor-containing composition in the lyophilized state according to the present embodiment may contain the anti-inflammatory cytokine IL-1Ra. The content of IL-1Ra is preferably 300 pg / g or more, more preferably 450 pg / g or more, and further preferably 600 pg / g or more. The upper limit varies depending on the individual difference of the mammal from which the blood was collected, but is, for example, 150,000 pg / g.

<Inflammatory cytokine>
The blood-derived growth factor-containing composition according to this embodiment may contain the inflammatory cytokines IL-1β, IL-6 and TNF-α. The contents of IL-1β and IL-6 are independently, preferably 30 pg / g or less, more preferably 28 pg / g or less, and further preferably 27 pg / g or less. The content of TNF-α (pg / 1 g of dried product) is preferably 60 pg / g or less, more preferably 57 pg / g or less, and further preferably 54 pg / g or less.

<Component ratio>
The blood-derived factor-containing composition in the lyophilized state according to the present embodiment preferably contains growth factors and anti-inflammatory cytokines in a well-balanced manner. Specifically, the mass ratio of the anti-inflammatory cytokine IL-1Ra based on the mass of the growth factor VEGF is preferably 1: 1 to 50, more preferably 1: 1 to 15, and even more preferably. It is 1: 1 to 10. In addition, the blood-derived growth factor-containing composition preferably contains a large amount of growth factors and hardly contains inflammatory cytokines. Specifically, the mass ratio of the inflammatory cytokine IL-1β based on the mass of the growth factor VEGF is preferably 1: 0.001 to 0.05, more preferably 1: 0.001 to 0. It is 03, and more preferably 1: 0.001 to 0.01.

As described above, according to the present embodiment, the blood-derived component including the buffy coat component can be easily recovered, the growth factor is dramatically increased, and the blood-derived component which has a wound healing and tissue regeneration effect is exhibited. Growth factor-containing compositions can be prepared.

The blood-derived factor-containing composition according to the present embodiment can be used for regenerative medicine and the like, and in particular, can be used for PRP therapy and the like.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

(Preparation of Examples 1 to 5)
Approximately 34 mL of whole blood was appropriately collected from 5 males in a blood collection tube containing an ACD solution (BD Vacutainer collection tube Japan Becton Dickinson Co., Ltd. # 364606). These were refrigerated at 4 ° C. overnight (about 16 hours) in a constant temperature bath. After storage, the first centrifugation treatment {1200 rpm (equivalent to 280 G), 10 minutes, 24 ° C.} was performed, and the collected volume ratio was 1: 0 based on the plasma (15 mL: total volume volume) as the supernatant. .4) was recovered. The plasma was subjected to a second centrifugation {2680 rpm (equivalent to 1400 G), 24 ° C. for 10 minutes}, and separated into pellets and a supernatant. After separation, the supernatant having a volume ratio of 1: 2 was left based on the volume of the pellet, and the other supernatant (plasma) was removed. The pellet was suspended by the remaining supernatant to obtain a suspension (platelet rich plasma).

The platelet-rich plasma was exposed to -60 ° C for 10 minutes, frozen, and then thawed by taking it out and allowing it to stand in a dry bath set at 37 ° C for 15 minutes. After thawing, 6.5 mL each of Lactated Ringer's solution (manufactured by Fuso Pharmaceutical Indus Corporation) was added and resuspended in order to stabilize platelet-rich plasma.

This was transferred to a syringe and filtered by a membrane filter having a pore size of 0.45 μm. By this filtering, platelet-rich plasma was separated into cells such as platelets remaining on the filter and platelet-rich plasma that had passed through the filter. The platelet-rich plasma was transferred to a vial, subjected to freezing treatment at −60 ° C., and cryopreserved at the same temperature for 24 hours.

Each vial was subjected to vacuum freeze-drying treatment using FDU-1110 (manufactured by Tokyo Rika Kikai Co., Ltd.) under the conditions of about 5 Pa and about −45 ° C. for about 16 hours. In this way, the samples of Examples 1 to 5 were obtained.

(Preparation of Examples 6 to 10)
Approximately 85 mL of whole blood was collected from one male into a blood collection tube containing an ACD solution and dispensed into 5 vessels. In this way, samples 6 to 10 were obtained. Then, the samples 7 and 8 were refrigerated in the refrigerator at 4 ° C. for about 24 hours and about 48 hours, respectively. The samples 9 and 10 were allowed to stand at room temperature (25 ° C.) for about 24 hours and about 48 hours, respectively. Similar to Examples 1 to 5, these samples 6 to 10 were subjected to a step of collecting platelet-rich plasma, a step of freeze-thawing and activating, and a step of freeze-drying to obtain samples of Examples 6 to 10. ..

(Measurement of each factor)
For the samples of Examples 1 to 5, 1 mL of water for injection was added to 300 mg of the dried product and thawed, and the amounts / concentrations of growth factors, anti-inflammatory cytokines, and inflammatory cytokines were analyzed by the ELISA method. The results are shown in Table 1. The contents of VEGF, IL-1Ra, IL-1β, IL-6 and TNF-α were 100 pg / mL or more, 100 pg / mL or more, 10 pg / mL or less, 10 pg / mL or less and 20 pg / mL or less, respectively.
Table 2 shows the results of the amount of growth factor, anti-inflammatory cytokine, and inflammatory cytokine / dried product (1 g) calculated from this.

Based on the above-mentioned analysis results for the samples of Examples 1 to 5, Table 3 shows the ratio of each cytokine amount based on the amount of growth factor VEGF.

(Confirmation of the effect of pre-refrigeration)
For the samples of Examples 6 to 10, 1 mL of water for injection was added to 300 mg of the dried product and thawed, and the amount / concentration of growth factor (VEGF) and anti-inflammatory cytokine (IL-1Ra) was analyzed by the ELISA method. FIG. 1 shows the ratio of each cytokine amount when 100% was prepared without pre-refrigeration after blood collection (Example 6). When pre-refrigerated at 4 ° C. for 24 hours (Example 7) or 48 hours (Example 8), the content of each factor increased to about 500% for IL-1Ra and about 200% for VEGF. On the other hand, when the mixture was allowed to stand at room temperature for 24 hours (Example 9) or 48 hours (Example 10) instead of pre-refrigeration, no significant increase in each factor was confirmed.

Claims (8)

A method for preparing a blood-derived growth factor-containing composition from mammalian blood.
The step of adding an anticoagulant capable of forming a chelate complex with a metal ion to the blood, and
A step of separating platelet-rich plasma containing a buffy coat component from the blood to which the anticoagulant is added, and
The step of freezing and thawing the platelet-rich plasma to activate it,
Including
The step of separating the platelet-rich plasma is
The step of recovering the upper layer containing plasma and the buffy coat by subjecting the blood to which the anticoagulant has been added to 100 to 1000 G, a first centrifugation treatment for 1 to 30 minutes; 1000 to 2500 G for the upper layer, A step of pelletizing platelets and a buffy coat component by performing a second centrifugation treatment for 1 to 30 minutes; and a step of removing the supernatant and suspending the platelets and the buffy coat component to obtain platelet-rich plasma. Including;
In the step of recovering the upper layer portion, the volume ratio of the upper layer portion to be recovered is 1: 0.1 to 0.5 and the volume ratio of the upper layer portion to be recovered is 1: 0.1 to 0.5 with respect to the volume of the total liquid amount.
In the step of obtaining platelet-rich plasma, a supernatant having a volume ratio of 1: 1 to 10 is left with reference to the volume of the pellet, and the rest is removed.
Method. The method of claim 1 , wherein the anticoagulant comprises citric acid, EDTA, or a salt thereof. The step of freeze-thawing and activating the platelet-rich plasma is performed by freezing by treatment at −200 ° C. to −20 ° C. for 10 minutes or longer, and then thawing by treatment at 20 ° C. to 50 ° C. for 10 minutes or longer. , The method according to claim 1 or 2 . The method according to any one of claims 1 to 3 , further comprising a step of pre-refrigerating the blood to which the anticoagulant is added at 0 to 10 ° C. before the step of separating the platelet-rich plasma. The method according to any one of claims 1 to 4 , further comprising a step of filtering the activated platelet-rich plasma using a filter having a pore size of 0.2 to 1 μm. The method according to any one of claims 1 to 5 , further comprising a step of freeze-drying the activated platelet-rich plasma. A blood-derived growth factor-containing composition prepared by the method according to any one of claims 1 to 5. A lyophilized product containing a blood-derived growth factor prepared by the method according to claim 6.

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