WO2014007422A1 - Composition for preserving platelets, kit for preserving platelets comprising the same, and method for preserving platelets using the same - Google Patents

Composition for preserving platelets, kit for preserving platelets comprising the same, and method for preserving platelets using the same Download PDF

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Publication number
WO2014007422A1
WO2014007422A1 PCT/KR2012/006319 KR2012006319W WO2014007422A1 WO 2014007422 A1 WO2014007422 A1 WO 2014007422A1 KR 2012006319 W KR2012006319 W KR 2012006319W WO 2014007422 A1 WO2014007422 A1 WO 2014007422A1
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WIPO (PCT)
Prior art keywords
platelets
preserving
composition
preserving platelets
chloride
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Application number
PCT/KR2012/006319
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French (fr)
Inventor
Hong-Seung Kim
Original Assignee
Hong-Seung Kim
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Publication of WO2014007422A1 publication Critical patent/WO2014007422A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture

Definitions

  • the present invention relates to a composition for preserving platelets, a kit for preserving platelets comprising the same, and a method for preserving platelets using the same, and more particularly, to a composition allowing for the preservation of platelets for an extended period of time while maintaining platelets in an activated state, a kit for preserving platelets comprising the same, and a method for preserving platelets using the same.
  • platelets are produced in bone marrow, and survive for about 7 days in vivo.
  • their storable period is shorter than that of other blood products and it is well known that platelets can currently only be stored for about 5 days in a shaking machine at room temperature.
  • An aspect of the present invention provides a composition for the long-term preservation of platelets.
  • Another aspect of the present invention provides a kit for the long-term preservation of platelets.
  • Another aspect of the present invention provides a method for preserving platelets using the composition and the kit.
  • composition for preserving platelets comprising adenosine triphosphate (ATP) , a divalent cation, a chloride, vitamin B, insulin, Granulocyte colony-stimulating factors (G-CSF) , erythropoietin, and a solvent.
  • ATP adenosine triphosphate
  • G-CSF Granulocyte colony-stimulating factors
  • the preserved platelets may be CD61 and von Willebrand factor (vWF) cell marker positive.
  • vWF von Willebrand factor
  • the divalent cation may be one or more selected from the group consisting of Zn 2+ , Cu 2+ , Mg 2+ , and Cr 2+ .
  • the chloride may be one or more selected from the group consisting of potassium chloride and bromine chloride .
  • the vitamin B may be vitamin B6 (pyridoxine) .
  • the solvent may be distilled water.
  • the composition for preserving platelets may comprise 15-25 mg of adenosine triphosphate (ATP) , 4-6 mg of the divalent cation, 100-200 mg of the chloride, 45-55 mg of vitamin B, 2.5-3.5 I.U. of insulin, 10-15 ⁇ of Granulocyte colony-stimulating factors (G-CSF) , 350-450 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc.
  • ATP adenosine triphosphate
  • G-CSF Granulocyte colony-stimulating factors
  • kits for preserving platelets comprising an airtight sterile container, which contains the above composition for preserving platelets, and in which internal pressure is controlled to be equal to external pressure thereof .
  • a method for preserving platelets comprising: mixing blood with an anticoagulant, centrifuging the blood, and obtaining a buffy coat and plasma; pouring the obtained buffy coat and plasma into an airtight sterile container, which contains the composition for preserving platelets, and in which internal pressure is controlled to be equal to external pressure thereof; and preserving the buffy coat and plasma poured container at a temperature from about 35 to about 37 °C.
  • the preserved platelets may be CD61 and von Willebrand factor (vWF) cell marker positive.
  • vWF von Willebrand factor
  • composition, the kit, and the method for preserving platelets of the present invention long-term preservation of platelets for at least 4 weeks or longer is possible, and during preservation, platelets can be maintained in an activated state.
  • the present invention can be used in various fields associated with research into platelet-related growth factors and the like.
  • FIGS. 1 to 3 illustrate results obtained by preserving platelets for 28 days under the same conditions using the kit for preserving platelets of the present invention, into which three groups of peripheral blood samples derived from different subjects were poured, upon which immunohistochemistry staining was carried out, and then peripheral blood samples were observed under a microscope (X400) .
  • FIGS. 1 to 3 illustrates the result of immunohistochemistry staining for CD61, and (b) illustrates the result of immunohistochemistry staining for v F.
  • a composition for long-term preservation of platelets According to the composition of the present invention, platelets particularly can be preserved for an extended period of time while maintaining their activated state.
  • platelets which are CD61 and von Willebrand factor (vWF) cell marker positive can be preserved for an extended period of time while maintaining their state, and the CD61 and von Willebrand factor (vWF) cell markers can be found specifically in activated platelets.
  • vWF von Willebrand factor
  • the CD61 is a cell marker associated with glycoprotein Ilia on the surface of platelets and is a standard marker of platelets .
  • the vWF is a receptor for a clotting factor in plasma, and a cell marker exhibiting that platelets maintain their activated state and can perform the aggregation function.
  • a platelet storage temperature the most important issue in preservation of platelets, is not met, structural lesions occur in platelet cell membrane and platelets lose the aggregation function, and in that case, vWF is not activated.
  • the preservation of platelets refers to the storage and/or maintenance of platelets, and may be used interchangeably, and particularly, refers to the preservation of platelets in an activated state.
  • the composition for preserving platelets of the present invention comprises adenosine triphosphate (ATP) , a divalent cation, a chloride, vitamin B, insulin, Granulocyte colony- stimulating factors (G-CSF) , erythropoietin, and a solvent .
  • ATP adenosine triphosphate
  • G-CSF Granulocyte colony- stimulating factors
  • erythropoietin erythropoietin
  • water may be used for the solvent, and preferably, distilled water may be used for the solvent .
  • Adenosine triphosphate is also known as ATP, and acts as an energy source for maintaining and preserving platelet functions .
  • the ATP may be comprised in an amount of 15 to 25 mg, and preferably, 18 to 22 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc . Where the ATP is comprised in an amount of less than 15 mg, there may be a problem in which a supply of an energy source required for maintenance of platelets may be lowered. Where the ATP is comprised in an amount of more than 25 mg, there is concern for an occurrence of cytotoxicity.
  • the divalent cation is a component designed to stabilize cell membranes and balance electrolytes in the composition for preserving platelets of the present invention, and may be one or more selected from the group consisting of Zn 2+ , Cu 2+ , Mg 2+ , and Cr 2+ , and preferably, Zn 2+ .
  • the divalent cation may be included as an appropriate salt known to the art, including sulfates, citrates, and the like, such as copper sulfate, magnesium sulfate, zinc sulfate, and the like.
  • each component may be used alone, or two or more components may be used in combination.
  • the divalent cation may be comprised in an amount of 4 to 6 mg, and preferably, 4.2 to 5.6 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc.
  • the chloride comprised in the present invention plays a role in stabilizing cations and maintaining a suitable concentration of the composition for preserving platelets.
  • the chloride may be one or more selected from the group consisting of potassium chloride and bromine chloride, and preferably, potassium chloride.
  • the chloride may be comprised in an amount of 100 to 200 mg, and preferably, 130 to 180 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc .
  • the chloride is comprised in an amount of less than 100 mg, there may be a problem in which the osmotic pressure is lowered too far and where the chloride is comprised in an amount of more than 200 mg, there may be a problem in which the osmotic pressure may be raised too high.
  • the osmotic pressure is lowered or raised by too much as above, it may have an adverse affect by which both the preservation of platelets and the preservation of platelets in an activated state may not be achieved smoothly.
  • the vitamin B may be vitamin B6 (pyridoxine) , and the vitamin B plays a role in maintaining the composition for stably preserving platelets through an antioxidant function.
  • the vitamin B may be comprised in an amount of 45 to 55 mg, and preferably, 48 to 52 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc.
  • vitamin B6 is comprised in an amount of less than 45 mg, there may be a problem with the maintenance of the stability of the composition for preserving platelets, and where vitamin B6 is comprised in an amount of more than 55 mg, there is concern for an occurrence of cytotoxicity.
  • Insulin plays a role in transducting signals associated with cell division of platelet-precursor cells in the composition for preserving platelets, and R (Regular) insulin may be used for the insulin.
  • the insulin may be comprised in an amount of 2.5 to 3.5 I.U., and preferably, 2.8 to 3.2 ⁇ . ⁇ , , based on the final volume of the composition for preserving platelets of the present invention being 8 cc.
  • the insulin is comprised in an amount of less than 2.5 I.U.
  • the transduction of signals associated with cell division of platelet-precursor cells may be deteriorated
  • the insulin is comprised in an amount of more than 3.5 I.U/, there is a possibility of an occurrence of apoptosis.
  • G-CSF Granulocyte colony-stimulating factors
  • the G-CSF may be comprised in an amount of 10 to 15 ⁇ , and preferably, 13 to 15 ⁇ g, based on the final volume of the composition for preserving platelets of the present invention being 8 cc .
  • the G-CSF is comprised in an amount of less than 10 ⁇ , there may be a problem in which the transduction of signals associated with cell division of platelet-precursor cells may be deteriorated, and where the G-CSF is comprised in an amount of more than 15 pg, there is a concern for an occurrence of apoptosis .
  • the erythropoietin plays a role in transducting signals associated with cell division of platelet-precursor cells, and recombinant erythropoietin may be used for the erythropoietin.
  • the erythropoietin may be comprised in an amount of 350 to 450 I. U., and preferably, 380 to 420 I . U. , based on the final volume of the composition for preserving platelets of the present invention being 8 cc .
  • the erythropoietin is comprised in an amount of less than 350 I.U.
  • the transduction of signals associated with cell division of platelet-precursor cells may be deteriorated
  • the erythropoietin is comprised in an amount of more than 450 I.U.
  • the composition for preserving platelets of the present invention may comprise 15-25 mg of adenosine triphosphate (ATP) , 4-6 mg of the divalent cation, 100-200 mg of the chloride, 45-55 mg of vitamin B, 2.5-3.5 I. U. of insulin, 10-15 iq of Granulocyte colony- stimulating factors (G-CSF) , 350-450 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc.
  • ATP adenosine triphosphate
  • G-CSF Granulocyte colony- stimulating factors
  • kits for preserving platelets comprises an airtight container containing the above described composition for preserving platelets of the present invention.
  • the kit for preserving platelets may comprise an airtight sterile container, in which internal pressure is controlled to be equal to external pressure thereof .
  • a container usable for the kit for preserving platelets of the present invention may be a sterilized airtight container.
  • Materials for the container are not particularly limited, and any container suitable for preserving platelets may be usable, and glass may be used for the material of the container.
  • any other appropriate container known to the art, which can safely contain the composition for preserving platelets may be used.
  • vials may be used for the container.
  • the container may contain the composition for preserving platelets of the present invention in an amount from about 7/50 to about 9/50 of the overall volume.
  • the method of the present invention uses the above composition and the kit for preserving platelets of the present invention, and more particularly, comprises mixing blood with an anticoagulant , centrifuging the blood, and obtaining a buffy coat and plasma; pouring the obtained buffy coat and plasma into the airtight sterile container, which contains the composition for preserving platelets of the present invention, and in which internal pressure is controlled to be equal to external pressure thereof; and preserving the buffy coat and plasma poured container at a temperature from about 35 to about 37°C.
  • composition for preserving platelets and the kit for preserving platelets comprising the same, used for the method of the present invention, are the same as described above .
  • the blood is mixed with an anticoagulant, followed by centrifuging to obtain a buffy coat and plasma.
  • the blood may be peripheral blood, obtained from the periphery of the human body.
  • the peripheral blood may be more preferably obtained from a vein.
  • the peripheral blood may be obtained from veins in arms, legs, or other extremities .
  • the usable anticoagulant may be, but is not limited to, one or more selected from the group of sodium citrate and heparin.
  • the anticoagulant may be mixed in a ratio of 6 to 7 parts by weight, and preferably, about 6.6 parts by weight, based on 100 parts by weight of peripheral blood. Where the anticoagulant is mixed in a ratio of less than 6 parts by weight based on 100 parts by weight of peripheral blood, the anticoagulant effect may be insufficient, and where the anticoagulant is mixed in a ratio of more than 7 parts by weight, there may be a problem in which citrate toxicity may occur.
  • the centrifugation for separating blood components may be carried out in a range of 1500 to 1700 rpm for 20 to 25 minutes, and more preferably, in a range of 1600 to 1700 rpm for 22 to 24 minutes.
  • centrifugation is carried out at less than 1500 rpm or for less than 20 minutes, there may be a problem in which the centrifugation may be carried out insufficiently. Where the centrifugation is carried out at more than 1700 rpm or for more than 25 minutes, there is a concern that cells may be damaged.
  • the bottom layer occupies about 45%, and is comprised of erythrocytes (red blood cells) .
  • the middle layer occupies about 1%, and is comprised of leukocytes (white blood cells) and platelets, and known as a 'buffy coat' .
  • the topmost layer occupies about 54%, and is comprised of plasma.
  • the buffy coat layer and the plasma layer isolated in the centrifugation detailed above are poured.
  • External pressure refers to atmospheric pressure, and the method for controlling the pressure may be carried out by, for example, penetrating a rubber stopper (rubber packing) with a syringe needle, followed by removing the syringe needle if the pressures are in equilibrium, but is not limited thereto.
  • the buffy coat layer and the plasma layer- poured container are preserved at a temperature from about 35 to about 37 °C, and more preferably, at a temperature of 36 °C.
  • the preservation is carried out at a temperature lower than 35 °C, there may be a problem in which the division of platelet-precursor cells may be deteriorated and the preservation efficiency of platelets in the composition for preserving platelets may be deteriorated.
  • the preservation is carried out at a temperature higher than 37 °C, there is a concern for an occurrence of cell damage from heat.
  • platelets can be preserved in an activated state for at least 28 days, that is, 4 weeks or more.
  • kit for preserving platelets comprising composition for preserving platelets
  • the mixed solution was centrifuged at 1670 rpm for 24 minutes, and from the separated blood, a buffy coat and plasma were collected with a pipette, and poured into a kit for preserving platelets, an airtight 50 cc glass bottle.
  • the composition for preserving platelets comprising the following components described in Table 1 was contained in the kit for preserving platelets, and distilled water was added as a solvent thereto to make up the final volume of the whole composition to
  • a 27 gauge syringe was inserted into a rubber stopper (rubber packing) on the airtight glass bottle, and removed if the pressures were in equilibrium to allow internal and external pressures to be equal.
  • each sample contained in each kit for preserving platelets was aspirated by a syringe and placed into a 10 mL test tube . Then, each tube was centrifuged at 3000 rpm for 10 minutes at room temperature, and pellets that sank to the bottom and were isolated were collected and embedded in paraffin to be fixed in 10% formalin solution. Cell blocks embedded in paraffin were formed, and then, to perform a hematoxylin-eosin staining, sections for each sample were prepared, and immunohistochemistry staining was carried out.
  • a list of antibodies in the immunofluorescent experiment for confirming the existence of platelets is CD61 and von Willebrand factor (vWF) . With the use of CD61 and vWF, it is possible to find platelets in an activated state.
  • CD61 and vWF von Willebrand factor
  • FIGS. 1 to 3 (a) illustrates the result of immunohistochemistry staining for CD61, and (b) illustrates the result of immunohistochemistry staining for vWF.

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Abstract

The present invention relates to a composition for preserving platelets, a kit for preserving platelets comprising the same, and a method for preserving platelets using the same, and more particularly, to a composition for long-term preservation of platelets for 4 weeks or longer, a kit for preserving platelets comprising the same, and a method for preserving platelets using the same.

Description

[DESCRIPTION]
[invention Title]
COMPOSITION FOR PRESERVING PLATELETS, KIT FOR PRESERVING PLATELETS COMPRISING THE SAME, AND METHOD FOR PRESERVING PLATELETS USING THE SAME
[Technical Field]
[0001] This application claims the priority of Korean Patent Application No. 10-2012-0073802 filed on July 06, 2012, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference .
[0002] The present invention relates to a composition for preserving platelets, a kit for preserving platelets comprising the same, and a method for preserving platelets using the same, and more particularly, to a composition allowing for the preservation of platelets for an extended period of time while maintaining platelets in an activated state, a kit for preserving platelets comprising the same, and a method for preserving platelets using the same.
[Background Art]
[0003] Interest in blood supply has been continuously increasing throughout the world. However, due to the relatively short stable storage period of blood products, storage periods (shelf-lives) for most concentrated erythrocytes, blood PCI/KR 2012. /DO 6319 products for blood transfusions, and the like, are currently restricted. After the restricted storage period, blood product pHs maybe greatly reduced, the level of ATP may also decrease rapidly, and the lifespan thereof may be greatly shortened in circulation, after a transfusion.
[0004] In particular, platelets are produced in bone marrow, and survive for about 7 days in vivo. However, after the collection of blood, their storable period is shorter than that of other blood products and it is well known that platelets can currently only be stored for about 5 days in a shaking machine at room temperature.
[0005] One reason why the storage stability of platelets is much lower than other blood products such as erythrocytes is that mitochondria exist in platelets, and metabolism by mitochondria occurs continuously therein. Thus, as the storage time passes, platelets exhibit a decline in pH along with a reduction in an ATP level, and entail the formation of lactates, but, the condition thereof tends to decline, due to the action of mitochondria.
[0006] A great deal of research into increasing the storage or preservation time of platelets has been carried out ; however, to date, platelets have not been successfully stored for one week or more . [Disclosure] [Technical Problem]
[0007] Platelets are one kind of blood corpuscle among blood corpuscles, components present within peripheral blood, and contribute to primary hemostasis through adhesion and aggregation. Platelets also produce many growth factors including platelet derived growth factor (PDF) . Thus, if a method of preserving platelets for an extended period of time and continuously maintaining their activated state was to be developed, it would be used extensively in research into related growth factors and commercially in various related industries .
[Technical Solution]
[0008] An aspect of the present invention provides a composition for the long-term preservation of platelets.
[0009] Another aspect of the present invention provides a kit for the long-term preservation of platelets.
[0010] Another aspect of the present invention provides a method for preserving platelets using the composition and the kit.
[0011] According to an aspect of the present invention, there is provided a composition for preserving platelets, comprising adenosine triphosphate (ATP) , a divalent cation, a chloride, vitamin B, insulin, Granulocyte colony-stimulating factors (G-CSF) , erythropoietin, and a solvent.
[0012] The preserved platelets may be CD61 and von Willebrand factor (vWF) cell marker positive.
[0013] The divalent cation may be one or more selected from the group consisting of Zn2+, Cu2+, Mg2+, and Cr2+.
[0014] The chloride may be one or more selected from the group consisting of potassium chloride and bromine chloride .
[0015] The vitamin B may be vitamin B6 (pyridoxine) .
[0016] The solvent may be distilled water.
[0017] The composition for preserving platelets may comprise 15-25 mg of adenosine triphosphate (ATP) , 4-6 mg of the divalent cation, 100-200 mg of the chloride, 45-55 mg of vitamin B, 2.5-3.5 I.U. of insulin, 10-15 μς of Granulocyte colony-stimulating factors (G-CSF) , 350-450 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc.
[0018] According to another aspect of the present invention, there is provided a kit for preserving platelets, comprising an airtight sterile container, which contains the above composition for preserving platelets, and in which internal pressure is controlled to be equal to external pressure thereof .
[0019] According to another aspect of the present invention, there is provided a method for preserving platelets, comprising: mixing blood with an anticoagulant, centrifuging the blood, and obtaining a buffy coat and plasma; pouring the obtained buffy coat and plasma into an airtight sterile container, which contains the composition for preserving platelets, and in which internal pressure is controlled to be equal to external pressure thereof; and preserving the buffy coat and plasma poured container at a temperature from about 35 to about 37 °C.
[0020] Preservation may be maintained for 28 days or longer.
[0021] The preserved platelets may be CD61 and von Willebrand factor (vWF) cell marker positive.
[Advantageous Effects]
[0022] With the composition, the kit, and the method for preserving platelets of the present invention, long-term preservation of platelets for at least 4 weeks or longer is possible, and during preservation, platelets can be maintained in an activated state. Thus, the present invention can be used in various fields associated with research into platelet-related growth factors and the like.
[Description of Drawings]
[0023] The above and other aspects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIGS. 1 to 3 illustrate results obtained by preserving platelets for 28 days under the same conditions using the kit for preserving platelets of the present invention, into which three groups of peripheral blood samples derived from different subjects were poured, upon which immunohistochemistry staining was carried out, and then peripheral blood samples were observed under a microscope (X400) .
In respective FIGS. 1 to 3, (a) illustrates the result of immunohistochemistry staining for CD61, and (b) illustrates the result of immunohistochemistry staining for v F.
[Best Mode]
[0024 ] Exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings .
[0025] According to the present invention, there is provided a composition for long-term preservation of platelets. According to the composition of the present invention, platelets particularly can be preserved for an extended period of time while maintaining their activated state.
[0026] According to the present invention, platelets which are CD61 and von Willebrand factor (vWF) cell marker positive can be preserved for an extended period of time while maintaining their state, and the CD61 and von Willebrand factor (vWF) cell markers can be found specifically in activated platelets.
[0027 ] The CD61 is a cell marker associated with glycoprotein Ilia on the surface of platelets and is a standard marker of platelets . The vWF is a receptor for a clotting factor in plasma, and a cell marker exhibiting that platelets maintain their activated state and can perform the aggregation function. When a platelet storage temperature, the most important issue in preservation of platelets, is not met, structural lesions occur in platelet cell membrane and platelets lose the aggregation function, and in that case, vWF is not activated.
[0028] In the present invention, "the preservation of platelets" refers to the storage and/or maintenance of platelets, and may be used interchangeably, and particularly, refers to the preservation of platelets in an activated state.
[0029] The composition for preserving platelets of the present invention comprises adenosine triphosphate (ATP) , a divalent cation, a chloride, vitamin B, insulin, Granulocyte colony- stimulating factors (G-CSF) , erythropoietin, and a solvent .
[0030] In the present invention, water may be used for the solvent, and preferably, distilled water may be used for the solvent .
[0031] Adenosine triphosphate is also known as ATP, and acts as an energy source for maintaining and preserving platelet functions .
[0032] The ATP may be comprised in an amount of 15 to 25 mg, and preferably, 18 to 22 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc . Where the ATP is comprised in an amount of less than 15 mg, there may be a problem in which a supply of an energy source required for maintenance of platelets may be lowered. Where the ATP is comprised in an amount of more than 25 mg, there is concern for an occurrence of cytotoxicity.
[0033] The divalent cation is a component designed to stabilize cell membranes and balance electrolytes in the composition for preserving platelets of the present invention, and may be one or more selected from the group consisting of Zn2+, Cu2+, Mg2+, and Cr2+, and preferably, Zn2+. The divalent cation may be included as an appropriate salt known to the art, including sulfates, citrates, and the like, such as copper sulfate, magnesium sulfate, zinc sulfate, and the like.
[ 0034] In the present invention, the term 'one or more' means that each component may be used alone, or two or more components may be used in combination.
[0035] The divalent cation may be comprised in an amount of 4 to 6 mg, and preferably, 4.2 to 5.6 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc.
[0036] Where the divalent cation is comprised in an amount of less than 4 mg, there may be a problem with cell membrane stability due to a lower osmotic pressure. Where the divalent cation is comprised in an amount of more than 6 mg, there is concern that cells may die due to high osmotic pressure in the composition for preserving platelets. [0037] The chloride comprised in the present invention plays a role in stabilizing cations and maintaining a suitable concentration of the composition for preserving platelets. The chloride may be one or more selected from the group consisting of potassium chloride and bromine chloride, and preferably, potassium chloride.
[0038] The chloride may be comprised in an amount of 100 to 200 mg, and preferably, 130 to 180 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc .
[0039] Where the chloride is comprised in an amount of less than 100 mg, there may be a problem in which the osmotic pressure is lowered too far and where the chloride is comprised in an amount of more than 200 mg, there may be a problem in which the osmotic pressure may be raised too high. When the osmotic pressure is lowered or raised by too much as above, it may have an adverse affect by which both the preservation of platelets and the preservation of platelets in an activated state may not be achieved smoothly.
[0040] The vitamin B may be vitamin B6 (pyridoxine) , and the vitamin B plays a role in maintaining the composition for stably preserving platelets through an antioxidant function.
[0041] The vitamin B may be comprised in an amount of 45 to 55 mg, and preferably, 48 to 52 mg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc.
[0042] Where vitamin B6 is comprised in an amount of less than 45 mg, there may be a problem with the maintenance of the stability of the composition for preserving platelets, and where vitamin B6 is comprised in an amount of more than 55 mg, there is concern for an occurrence of cytotoxicity.
[0043 ] Insulin plays a role in transducting signals associated with cell division of platelet-precursor cells in the composition for preserving platelets, and R (Regular) insulin may be used for the insulin.
[0044] The insulin may be comprised in an amount of 2.5 to 3.5 I.U., and preferably, 2.8 to 3.2 Ι.ϋ, , based on the final volume of the composition for preserving platelets of the present invention being 8 cc.
[0045] Where the insulin is comprised in an amount of less than 2.5 I.U. , there may be a problem in which the transduction of signals associated with cell division of platelet-precursor cells may be deteriorated, and where the insulin is comprised in an amount of more than 3.5 I.U/, there is a possibility of an occurrence of apoptosis.
[ 0046] Granulocyte colony-stimulating factors (G-CSF) , also known as filgrastim, play a role in transducting signals associated with cell division of platelet-precursor cells, and recombinant G-CSF may be used for the G-CSF.
[0047 ] The G-CSF may be comprised in an amount of 10 to 15 μς, and preferably, 13 to 15 μg, based on the final volume of the composition for preserving platelets of the present invention being 8 cc .
[0048] Where the G-CSF is comprised in an amount of less than 10 μς, there may be a problem in which the transduction of signals associated with cell division of platelet-precursor cells may be deteriorated, and where the G-CSF is comprised in an amount of more than 15 pg, there is a concern for an occurrence of apoptosis .
[0049] The erythropoietin plays a role in transducting signals associated with cell division of platelet-precursor cells, and recombinant erythropoietin may be used for the erythropoietin.
[0050] The erythropoietin may be comprised in an amount of 350 to 450 I. U., and preferably, 380 to 420 I . U. , based on the final volume of the composition for preserving platelets of the present invention being 8 cc .
[0051] Where the erythropoietin is comprised in an amount of less than 350 I.U., there may be a problem in which the transduction of signals associated with cell division of platelet-precursor cells may be deteriorated, and where the erythropoietin is comprised in an amount of more than 450 I.U. , there is a concern for an occurrence of apoptosis.
[0052] Most preferably, the composition for preserving platelets of the present invention may comprise 15-25 mg of adenosine triphosphate (ATP) , 4-6 mg of the divalent cation, 100-200 mg of the chloride, 45-55 mg of vitamin B, 2.5-3.5 I. U. of insulin, 10-15 iq of Granulocyte colony- stimulating factors (G-CSF) , 350-450 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc.
[0053] Meanwhile, according to the present invention, there is provided a kit for preserving platelets . The kit for preserving platelets of the present invention comprises an airtight container containing the above described composition for preserving platelets of the present invention.
[0054] Furthermore, the kit for preserving platelets may comprise an airtight sterile container, in which internal pressure is controlled to be equal to external pressure thereof .
[0055] A container usable for the kit for preserving platelets of the present invention may be a sterilized airtight container. Materials for the container are not particularly limited, and any container suitable for preserving platelets may be usable, and glass may be used for the material of the container. However, any other appropriate container known to the art, which can safely contain the composition for preserving platelets, may be used. Most preferably, vials may be used for the container.
[0056] The container may contain the composition for preserving platelets of the present invention in an amount from about 7/50 to about 9/50 of the overall volume.
[0057 ] Furthermore, according to the present invention, there is provided a method for preserving platelets for an extended period of time .
[0058] The method of the present invention uses the above composition and the kit for preserving platelets of the present invention, and more particularly, comprises mixing blood with an anticoagulant , centrifuging the blood, and obtaining a buffy coat and plasma; pouring the obtained buffy coat and plasma into the airtight sterile container, which contains the composition for preserving platelets of the present invention, and in which internal pressure is controlled to be equal to external pressure thereof; and preserving the buffy coat and plasma poured container at a temperature from about 35 to about 37°C.
[0059] The composition for preserving platelets and the kit for preserving platelets comprising the same, used for the method of the present invention, are the same as described above .
[0060] More particularly, for the method for preserving platelets of the present invention, first, in order to prevent the coagulation of blood obtained from humans, the blood is mixed with an anticoagulant, followed by centrifuging to obtain a buffy coat and plasma.
[0061] Here, the blood may be peripheral blood, obtained from the periphery of the human body. The peripheral blood may be more preferably obtained from a vein. For example, the peripheral blood may be obtained from veins in arms, legs, or other extremities . [0062] Here, the usable anticoagulant may be, but is not limited to, one or more selected from the group of sodium citrate and heparin.
[0063] The anticoagulant may be mixed in a ratio of 6 to 7 parts by weight, and preferably, about 6.6 parts by weight, based on 100 parts by weight of peripheral blood. Where the anticoagulant is mixed in a ratio of less than 6 parts by weight based on 100 parts by weight of peripheral blood, the anticoagulant effect may be insufficient, and where the anticoagulant is mixed in a ratio of more than 7 parts by weight, there may be a problem in which citrate toxicity may occur.
[0064] The centrifugation for separating blood components may be carried out in a range of 1500 to 1700 rpm for 20 to 25 minutes, and more preferably, in a range of 1600 to 1700 rpm for 22 to 24 minutes.
[0065] Where the centrifugation is carried out at less than 1500 rpm or for less than 20 minutes, there may be a problem in which the centrifugation may be carried out insufficiently. Where the centrifugation is carried out at more than 1700 rpm or for more than 25 minutes, there is a concern that cells may be damaged.
[0066] Once the centrifugation is completed, blood is divided into three layers. The bottom layer occupies about 45%, and is comprised of erythrocytes (red blood cells) . The middle layer occupies about 1%, and is comprised of leukocytes (white blood cells) and platelets, and known as a 'buffy coat' . The topmost layer occupies about 54%, and is comprised of plasma.
[0067] Into the airtight sterile container, which contains the composition for preserving platelets of the present invention, and in which of internal pressure is controlled to be equal to external pressure thereof, the buffy coat layer and the plasma layer isolated in the centrifugation detailed above are poured. External pressure refers to atmospheric pressure, and the method for controlling the pressure may be carried out by, for example, penetrating a rubber stopper (rubber packing) with a syringe needle, followed by removing the syringe needle if the pressures are in equilibrium, but is not limited thereto.
[0068] Subsequently, the buffy coat layer and the plasma layer- poured container are preserved at a temperature from about 35 to about 37 °C, and more preferably, at a temperature of 36 °C. Where the preservation is carried out at a temperature lower than 35 °C, there may be a problem in which the division of platelet-precursor cells may be deteriorated and the preservation efficiency of platelets in the composition for preserving platelets may be deteriorated. Where the preservation is carried out at a temperature higher than 37 °C, there is a concern for an occurrence of cell damage from heat.
[0069] According to the method for preserving platelets, platelets can be preserved in an activated state for at least 28 days, that is, 4 weeks or more. [0070] Hereinafter, the present invention will be described in more detail with reference to specific embodiments . However, the following embodiments are described for illustrative purposes only, and do not limit the scope of the present invention.
[Mode for Invention]
[0071] Embodiments
[0072] <Embodimenta>
[0073] 1. Preparation of kit for preserving platelets comprising composition for preserving platelets
[0074] To prevent coagulation, 30 cc of blood extracted from a vein was mixed with 2 cc of a citrate buffer (2.45 g of dextrose, 2.2 g of sodium citrate, and 730 mg citrate are contained in 100 cc of the citrate buffer) (Fenwal) to prepare a mixed solution.
[0075] The mixed solution was centrifuged at 1670 rpm for 24 minutes, and from the separated blood, a buffy coat and plasma were collected with a pipette, and poured into a kit for preserving platelets, an airtight 50 cc glass bottle.
[0076] For the buffy coat and plasma obtained by the centrifugation of 30 cc of blood, the composition for preserving platelets comprising the following components described in Table 1 was contained in the kit for preserving platelets, and distilled water was added as a solvent thereto to make up the final volume of the whole composition to
[0077] [Table l]
Figure imgf000018_0001
[0078] A 27 gauge syringe was inserted into a rubber stopper (rubber packing) on the airtight glass bottle, and removed if the pressures were in equilibrium to allow internal and external pressures to be equal.
[0079] 2. Preservation process of platelets
[0080] Blood samples were collected from three volunteers. Three groups of the kits for preserving platelets as prepared in "1." were formed and incubated for 28 days under the same conditions. The incubation temperature was 36 °C. [0081] 3. Confirmation of results for platelet preservation [0082] (1) Immunohistochemistry staining
[0083] After preservation for 28 days, each sample contained in each kit for preserving platelets was aspirated by a syringe and placed into a 10 mL test tube . Then, each tube was centrifuged at 3000 rpm for 10 minutes at room temperature, and pellets that sank to the bottom and were isolated were collected and embedded in paraffin to be fixed in 10% formalin solution. Cell blocks embedded in paraffin were formed, and then, to perform a hematoxylin-eosin staining, sections for each sample were prepared, and immunohistochemistry staining was carried out.
[0084] Immunostaining for CD61 and von Willebrand factor (vWF) was carried out passively, and Dako Catalyzed Signal Amplification (Dako) was applied. After deparaffinization and blocking of endogenous peroxidase activity, sections were treated with protein block solution for 5 minutes, and then, treated with antibodies (CD61 and von Willebrand factor (vWF) ) for 20 minutes.
[0085] As described above, after the immunohistochemistry staining, observation was made under a microscope (X400) . The results for a first, second, and third group are shown in FIG. 1, FIG. 2, and FIG. 3, respectively.
[0086] A list of antibodies in the immunofluorescent experiment for confirming the existence of platelets is CD61 and von Willebrand factor (vWF) . With the use of CD61 and vWF, it is possible to find platelets in an activated state.
[0087] Markers for immunohistochemistry staining used in this experiment were as per the following Table 2.
[0088] [Table 2]
Figure imgf000020_0001
[0089] In FIGS. 1 to 3 , (a) illustrates the result of immunohistochemistry staining for CD61, and (b) illustrates the result of immunohistochemistry staining for vWF.
[0090] (2) Conclusion
[0091] Referring to FIGS. 1 to 3, after 28 days of platelet preservation, platelets in an activated state were maintained in large amounts, and they exhibited strong immunoreactivity for both CD61 and von Willebrand factor (vWF) .
[0092] As shown in the result of the above experiment, it can be found that activated platelets could be preserved up to 28 days, and furthermore, experiments for all three groups showed the same results .
[0093] That is, this result confirms that the long-term preservation of platelets for 4 weeks or longer was possible with the use of the composition, the kit, and the method for preserving platelets of the present invention. [0094] While the present invention has been shown and described in connection with the exemplary embodiments, it will be apparent to those skilled in the art that modifications and variations can be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

[CLAIMS]
[Claim l]
A composition for preserving platelets, comprising adenosine triphosphate (ATP) , a divalent cation, a chloride, vitamin B, insulin, Granulocyte colony-stimulating factors (G-CSF) , erythropoietin, and a solvent.
[Claim 2]
The composition for preserving platelets of claim 1, wherein the preserved platelets are CD61 and von Willebrand factor (vWF) cell marker positive.
[Claim 3]
The composition for preserving platelets of claim 1, wherein the divalent cation is one or more selected from the group consisting of Zn+, Cu2+, Mg2+, and Cr2+.
[Claim 4]
The composition for preserving platelets of claim 1, wherein the chloride is one or more selected from the group consisting of potassium chloride and bromine chloride.
[Claim 5]
The composition for preserving platelets of claim 1, wherein the vitamin B is vitamin B6 (pyridoxine) .
[Claim 6]
The composition for preserving platelets of claim 1, wherein the solvent is distilled water.
[Claim 7]
The composition for preserving platelets of claim 1, comprising 15-25 mg of adenosine triphosphate (ATP) , 4-6 mg of the divalent cation, 100-200 mg of the chloride, 45-55 mg of vitamin B, 2.5-3.5 I.U. of insulin, 10-15 g of Granulocyte colony-stimulating factors (G-CSF) , 350-450 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc .
[Claim 8]
A kit for preserving platelets, comprising an airtight sterile container, which contains the composition for preserving platelets of any of claims 1 to 7, and in which internal pressure is controlled to be equal to external pressure thereof .
[Claim 9]
A method for preserving platelets, comprising:
mixing blood with an anticoagulant, centrifuging the blood, and obtaining a buffy coat and plasma; pouring the obtained buffy coat and plasma into an airtight sterile container, which contains the composition for preserving platelets of any of claims 1 to 7, and in which internal pressure is controlled to be equal to external pressure thereof; and
preserving the buffy coat and plasma poured container at a temperature from about 35 to about 37 °C.
[Claim 10]
The method for preserving platelets of claim 9, wherein preservation is maintained for 28 days or longer.
[Claim ll]
The method for preserving platelets of claim 9, wherein the preserved platelets are CD61 and von Willebrand factor (vWF) cell marker positive.
PCT/KR2012/006319 2012-07-06 2012-08-08 Composition for preserving platelets, kit for preserving platelets comprising the same, and method for preserving platelets using the same WO2014007422A1 (en)

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WO2007132718A1 (en) * 2006-05-12 2007-11-22 Kowa Company, Ltd. Solid preparation comprising adenosine 5'-triphosphate or physiologically acceptable salt thereof
US20090029342A1 (en) * 2004-06-29 2009-01-29 Cheng-Yao Su Medium and method for preserving platelets, red blood cells, and other non-nucleus cells and platelets-containing composition
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US20090029342A1 (en) * 2004-06-29 2009-01-29 Cheng-Yao Su Medium and method for preserving platelets, red blood cells, and other non-nucleus cells and platelets-containing composition
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US20160205923A1 (en) * 2015-01-21 2016-07-21 Hong Seung Kim Activated platelet preservation composition, method for preserving activated platelet and preserved activated platelet using the same
US10015959B2 (en) * 2015-01-21 2018-07-10 Hong Seung Kim Activated platelet preservation composition, method for preserving activated platelet and preserved activated platelet using the same
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