WO2014007421A1 - Culture composition for inducing activated t lymphocytes from peripheral blood, culture kit comprising the same, and method for inducing activated t lymphocytes from peripheral blood using the same - Google Patents

Culture composition for inducing activated t lymphocytes from peripheral blood, culture kit comprising the same, and method for inducing activated t lymphocytes from peripheral blood using the same Download PDF

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Publication number
WO2014007421A1
WO2014007421A1 PCT/KR2012/006318 KR2012006318W WO2014007421A1 WO 2014007421 A1 WO2014007421 A1 WO 2014007421A1 KR 2012006318 W KR2012006318 W KR 2012006318W WO 2014007421 A1 WO2014007421 A1 WO 2014007421A1
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Prior art keywords
lymphocytes
culture composition
peripheral blood
culture
activated
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PCT/KR2012/006318
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French (fr)
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Hong-Seung Kim
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Hong-Seung Kim
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin

Definitions

  • the present invention relates to a culture composition for inducing activated T lymphocytes from peripheral blood, a culture kit comprising the same, and a method for inducing activated T lymphocytes from peripheral blood using the same, and more particularly, to a culture composition for inducing CD3 and CD25 positive T cells by the directed differentiation of leukocytes obtained from peripheral blood, a culture kit comprising the same, and a method for inducing CD3 and CD25 positive T cells using the same .
  • T cells also known as T lymphocytes, are derived from hematopoietic stem cells in bone marrow, differentiate in the thymus and migrate to peripheral lymphatic tissues . T cells are distributed in the spleen and all parts of the thymus in lymph nodes .
  • T cells occupy 60-70% of lymphocytes in tissues or blood and the number of T cells is commonly slightly greater than that of B cells. While T cells cannot be morphologically distinguished from B cells, they can be identified and isolated by differences in cell surface antigens (CD4, CD8, CD3, or Thyl antigens in mice, and the like) .
  • T cells are classified largely into CD4 + CD8 " T cells and CD4 " CD8 + T cells according to antigenic CD4 and CD8 molecules expressed on their surface.
  • T cells are divided into several subgroups having different functions, including helper T cells and cells mediating delayed type hypersensitivity, belonging to the former, and killer T cells belonging to the latter, and the like.
  • CD3 is a cell marker which is commonly expressed in all T lymphocytes and proves that unknown cells are T cells.
  • CD25-expressing T cells are considered to be activated T lymphocytes (T cell subsets: A synopsis of the lecture by Dr. Steve Cobbold for the FHS Physiology Immunology Option 1/ Definitions and relationships of different T cell subsets http: //users .path. ox. ac . uk/ ⁇ scobbold/Teaching/TSUBSETS .pdf) .
  • CD25-positive cells are activated T lymphocytes, and are known to have therapeutic effects on autoimmune diseases and tumors (Romagnoli Paola, Tellier Julie, Van Meerwijik Joost Pm: Genetic control of thymic development of CD4+, CD25+FoxP3+ regulatory T lymphocytes, Eur J Immunol, 2005 Dec; 35 (12) :3525-32) .
  • T cells are a major type of cells mediating cellular immunity against cancer cells, viruses, or transplanted tissues or cells. In order to attack targets, T cells should be activated from a resting and nonproliferative state to an active and proliferatable state.
  • hematopoiesis in the human body is a one-way process of differentiation from multipotential hematopoietic stem cells to functional mature differentiated cells, such as granulocytes, monocytes, lymphocytes, and the like, as shown in FIG. 1.
  • An aspect of the present invention provides a culture composition for inducing activated T lymphocytes from peripheral blood.
  • Another aspect of the present invention provides a culture kit for inducing activated T lymphocytes from peripheral blood. [0012] Another aspect of the present invention provides a method for inducing activated T lymphocytes using the culture composition and the culture kit.
  • a culture composition for inducing activated T lymphocytes from peripheral blood 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
  • erythropoietin erythropoietin
  • the induced T cells may be CD3 and CD25 cell marker positive .
  • 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.
  • the solvent may be distilled water.
  • the culture composition may comprise 10-15 mg of adenosine triphosphate (ATP) , 5-7 mg of the divalent cation, 80-100 mg of the chloride, 45-55 mg of vitamin B, 3-7 I.U. of insulin, 5-15 g of Granulocyte colony-stimulating factors (G-CSF), 5-15 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
  • erythropoietin erythropoietin
  • a culture kit for inducing activated T lymphocytes from peripheral blood comprising an airtight sterile container, which contains the above culture composition, and in which internal pressure is controlled to be equal to external pressure thereof.
  • a method for inducing activated T lymphocytes from peripheral blood 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 culture composition, and in which internal pressure is controlled to be equal to external pressure thereof; and inserting the buffy coat and plasma poured container into a low temperature incubator and incubating the buffy coat and plasma.
  • the incubation may be carried out for a period of f om about 50 hours to about 200 hours.
  • the incubation may be carried out at a temperature from about 37 to 39 °C.
  • the induced T cells may be CD3 and CD25 cell marker positive .
  • the culture kit of the present invention for inducing activated T lymphocytes from peripheral blood and the method for inducing activated T lymphocytes using the same, a large amount of activated T lymphocytes can easily be obtained from peripheral blood obtained from adults.
  • the obtained activated T lymphocytes can be used in various fields associated with the treatment of autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus, and in cell therapy products which can induce the reduction and destruction of tumors, and the like.
  • FIG. 1 is a diagram illustrating the differentiation process from hematopoietic stem cells to mature blood cells
  • FIG. 2(a) illustrates the culture composition of the present invention
  • FIG. 2(b) illustrates the result directly after pouring the buffy coat and plasma into the culture composition of the present invention
  • FIG. 2(c) illustrates the result after incubating the culture kit of the present invention for 4 weeks
  • FIGS. 3 to 5 illustrate results obtained by incubating peripheral blood samples for 28 days under the same conditions using the culture kit of the present invention, into which three groups of peripheral blood samples were poured, immunohistochemistry staining was carried out, and then peripheral blood samples were observed under a microscope (X400) .
  • FIGS. 3 to 5 illustrate results obtained by incubating peripheral blood samples for 28 days under the same conditions using the culture kit of the present invention, into which three groups of peripheral blood samples were poured, immunohistochemistry staining was carried out, and then peripheral blood samples were observed under a microscope (X400) .
  • FIGS. 3 to 5 illustrate results obtained by incubating peripheral blood samples for 28 days under the same conditions using the culture kit of the present invention, into which three groups of peripheral blood samples were poured, immunohistochemistry staining was carried out, and then peripheral blood samples were observed under a microscope (X400) .
  • FIGS. 3 to 5 illustrates the result of immunohistochemistry staining for CD3, and
  • FIGb illustrates the result of immuno
  • T cells can be obtained from differentiation-completed blood corpuscles in peripheral blood, in particular, leukocytes, by induction.
  • activated T lymphocytes which are CD3 and CD25 positive can be obtained from leukocytes obtained from peripheral blood.
  • CD3 is a cell marker which is commonly expressed in all T lymphocytes and proves that unknown cells are T cells, and among these T cells, CD25 -expressing T cells are considered to be activated T lymphocytes.
  • activated T lymphocytes can be induced from a buffy coat isolated from peripheral blood, and especially, activated T lymphocytes can be induced from leukocytes contained in the buffy coat.
  • the culture composition 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
  • 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 functions as an energy source for facilitating and maintaining the differentiation into activated T lymphocytes in the culture composition.
  • the ATP may be comprised in an amount of 10 to 15 mg, and preferably, 11 to 13 mg, based on the final volume of the culture composition of the present invention being 8 cc. Where the ATP is comprised in an amount of less than 10 mg, there may be a problem in which the directed incubation into activated T lymphocytes may not be carried out sufficiently, and where the ATP is comprised in an amount of more than 15 mg, there may be a problem in which the blood corpuscles may differentiate into a type of cell other than activated T lymphocytes .
  • the divalent cation is a component designed to stabilize cell membrane and balance electrolytes in the culture composition of the present invention, and may be one or more selected from the group consisting of Zn + , Cu 2+ , g 2+ , and Cr 2+ , and preferably, Zn + .
  • 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 5 to 7 mg, and preferably, 5.2 to 6.6 mg, based on the final volume of the culture composition of the present invention being 8 cc.
  • the divalent cation is comprised in an amount of less than 5 mg, there may be a problem in which the directed differentiation into activated T lymphocytes may be insufficient. Where the divalent cation is comprised in an amount of more than 7 mg, there is a concern that cells may die due to high osmotic pressure in a culture medium.
  • the chloride comprised in the present invention plays a role in stabilizing cations and maintaining a suitable concentration of a culture medium in the culture composition.
  • 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 170 mg, based on the final volume of the culture composition 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 is raised too high.
  • the osmotic pressure is lowered or raised by too much as above, it may have adverse affects on a cell culture, and the induction of activated T lymphocytes may not be achieved smoothly.
  • the vitamin B may be vitamin B6 (pyridoxine) , and the vitamin B acts as a catalyst for cell division in the culture composition.
  • 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 culture composition of the present invention being 8 cc.
  • vitamin B6 is comprised in an amount of less than 45 mg, there is a problem with the efficiency of cell division and differentiation, and where vitamin B6 is comprised in an amount of more than 55 mg, there is a concern for an occurrence of cytotoxicity.
  • Insulin acts as a messenger for cell differentiation in the culture composition, and R (Regular) insulin may be used for the insulin.
  • R Regular insulin
  • the insulin may be comprised in an amount of 3 to 7 I.U., and preferably, 4 to 6 I.U. , based on the final volume of the culture composition of the present invention being 8 cc.
  • the insulin is comprised in an amount of less than 3 I.U., there may be a problem in which the efficiency of cell division and differentiation may be deteriorated, and where the insulin is comprised in an amount of more than 7 I.U., there is a possibility of an occurrence of cell overproliferation and apoptosis.
  • G-CSF Granulocyte colony-stimulating factors
  • filgrastim act as a messenger for inducing differentiation in the cell differentiation process, and recombinant G-CSF may be used for the G-CSF.
  • the G-CSF may be comprised in an amount of 5 to 15 ⁇ ig, and preferably, 8 to 12 ⁇ , based on the final volume of the culture composition of the present invention being 8 cc.
  • the G-CSF is comprised in an amount of less than 5 g, there may be a problem in which the efficiency of cell division and differentiation may be deteriorated, and where the G-CSF is comprised in an amount of more than 15 pg, there is concern for an occurrence of cell overproliferation and apoptosis.
  • the erythropoietin also plays a role in inducing differentiation in the cell differentiation process, and recombinant erythropoietin may be used for the erythropoietin.
  • the erythropoietin may be comprised in an amount of 5 to 15 I.U., and preferably, 8 to 12 I.U., based on the final volume of the culture composition of the present invention being 8 cc.
  • the erythropoietin is comprised in an amount of less than 5 I .U.
  • the efficiency of cell division and differentiation may be deteriorated
  • the erythropoietin is comprised in an amount of more than 15 I .U.
  • the culture composition of the present invention may comprise 10-15 mg of adenosine triphosphate (ATP) , 5-7 mg of the divalent cation, 80-100 mg of the chloride, 45-55 mg of vitamin B, 3-7 I.U. of insulin, 5-15 ⁇ of Granulocyte colony-stimulating factors (G-CSF) , 5-15 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
  • the culture kit of the present invention comprises an airtight container containing the above described culture composition of the present invention.
  • the culture kit may comprise an airtight sterile container, in which internal pressure is controlled to be equal to external pressure thereof .
  • a container usable for the culture kit of the present invention may be a sterilized airtight container.
  • Materials for the container are not particularly limited, and any container suitable for culture growth 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 culture composition and allow cells to be cultured, may be used.
  • Most preferably, vials may be used for the container.
  • the container may contain the culture composition 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 culture composition and the culture kit of the present invention, and more particularly, comprises mixing peripheral 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 culture composition of the present invention, and in which internal pressure is controlled to be equal to external pressure thereof; and inserting the buffy coat and plasma poured container into a low temperature incubator and incubating the buffy coat and plasma.
  • the culture composition and the culture kit comprising the same, which are used for the method for inducing activated T lymphocytes from peripheral blood 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, which is 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 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 x 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 syringe needle into a rubber stopper (rubber packing) , 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 incubated in a low temperature incubator.
  • the low temperature incubation may be carried out for 50 to 200 hours, and preferably, for 70 to 160 hours . Where the incubation is carried out for less than 50 hours, there may be a problem in which the incubation may be insufficient. Where the incubation is carried out for more than 200 hours, there may be a problem in which cell concentration may increase excessively in the culture composition and thus, apoptosis or cell suicide may occur.
  • the incubation may be carried out at a temperature from 37 to 39 °C, and more preferably, at about 38 °C. Where the incubation is carried out at a temperature less than 37 °C, there may be a problem in which the blood corpuscles may differentiate into a type of cell other than activated T lymphocytes. Where the incubation is carried out at more than 39 °C, there is a concern of an occurrence of cell damage from heat.
  • 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 culture kit, an airtight 50 cc glass bottle.
  • the culture composition comprising the following components described in Table 1 was contained in the culture kit, and distilled water was added as a solvent thereto to make up the final volume of the whole composition to 8 cc.
  • 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 .
  • FIG. 2(a) illustrates the culture composition of the present invention
  • FIG. 2(b) illustrates the result directly after pouring the buffy coat and plasma into the culture composition of the present invention
  • FIG. 2(c) illustrates the result after incubating the culture kit of the present invention for 4 weeks.
  • each sample contained in each culture kit 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. Bond-MAX (Leica) equipment was used in immunohistochemistry staining.
  • a list of antibodies in the immunofluorescent experiment for confirming the cell differentiation into activated T lymphocytes is CD3 and CD25. Markers for immunohistochemistry staining used in this experiment were as per the following Table 2.
  • FIGS. 3 to 5 (a) illustrates the result of immunohistochemistry staining for CD3, and (b) illustrates the result of immunohistochemistry staining for CD25.
  • lymphoid cell marker CD3 was expressed and CD25 was also expressed. This indicates that activated T lymphocytes are obtained by the present invention. Experiment for three groups showed the same results .

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Abstract

The present invention relates to a culture composition for inducing T cells from peripheral blood, a culture kit comprising the same, and a method for inducing T cells from peripheral blood using the same, and more particularly, to a culture composition for inducing activated T lymphocytes from peripheral blood, comprising adenosine triphosphate (ATP), a divalent cation, a chloride, vitamin B6, insulin, Granulocyte colony-stimulating factors (G-CSF), erythropoietin, and a solvent, a culture kit comprising the same, and a method for inducing activated T lymphocytes from peripheral blood using the same.

Description

[DESCRIPTION]
[invention Title]
CULTURE COMPOSITION FOR INDUCING ACTIVATED T LYMPHOCYTES FROM PERIPHERAL BLOOD, CULTURE KIT COMPRISING THE SAME, AND METHOD FOR INDUCING ACTIVATED T LYMPHOCYTES FROM PERIPHERAL BLOOD USING THE SAME
[Technical Field]
[0001] This application claims the priority of Korean Patent Application No. 10-2012-0073803 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 culture composition for inducing activated T lymphocytes from peripheral blood, a culture kit comprising the same, and a method for inducing activated T lymphocytes from peripheral blood using the same, and more particularly, to a culture composition for inducing CD3 and CD25 positive T cells by the directed differentiation of leukocytes obtained from peripheral blood, a culture kit comprising the same, and a method for inducing CD3 and CD25 positive T cells using the same .
[Background Art]
[0003] T cells, also known as T lymphocytes, are derived from hematopoietic stem cells in bone marrow, differentiate in the thymus and migrate to peripheral lymphatic tissues . T cells are distributed in the spleen and all parts of the thymus in lymph nodes .
[0004] T cells occupy 60-70% of lymphocytes in tissues or blood and the number of T cells is commonly slightly greater than that of B cells. While T cells cannot be morphologically distinguished from B cells, they can be identified and isolated by differences in cell surface antigens (CD4, CD8, CD3, or Thyl antigens in mice, and the like) .
[0005] T cells are classified largely into CD4+CD8" T cells and CD4"CD8+ T cells according to antigenic CD4 and CD8 molecules expressed on their surface. T cells are divided into several subgroups having different functions, including helper T cells and cells mediating delayed type hypersensitivity, belonging to the former, and killer T cells belonging to the latter, and the like.
[0006] CD3 is a cell marker which is commonly expressed in all T lymphocytes and proves that unknown cells are T cells. Among these T cells, CD25-expressing T cells are considered to be activated T lymphocytes (T cell subsets: A synopsis of the lecture by Dr. Steve Cobbold for the FHS Physiology Immunology Option 1/ Definitions and relationships of different T cell subsets http: //users .path. ox. ac . uk/~scobbold/Teaching/TSUBSETS .pdf) .
Meanwhile, CD25-positive cells are activated T lymphocytes, and are known to have therapeutic effects on autoimmune diseases and tumors (Romagnoli Paola, Tellier Julie, Van Meerwijik Joost Pm: Genetic control of thymic development of CD4+, CD25+FoxP3+ regulatory T lymphocytes, Eur J Immunol, 2005 Dec; 35 (12) :3525-32) .
[0007] T cells (T-Lymphocytes) are a major type of cells mediating cellular immunity against cancer cells, viruses, or transplanted tissues or cells. In order to attack targets, T cells should be activated from a resting and nonproliferative state to an active and proliferatable state.
[0008] Meanwhile, hematopoiesis in the human body is a one-way process of differentiation from multipotential hematopoietic stem cells to functional mature differentiated cells, such as granulocytes, monocytes, lymphocytes, and the like, as shown in FIG. 1. [Disclosure]
[Technical Problem]
[0009] Thus, if it is possible to direct differentiation-completed peripheral blood, in particular, mature leukocytes, to differentiate activated T lymphocytes, it is expected that after directed incubation of activated T lymphocytes in vitro has been successfully undertaken, research and clinical applications undertaken with activated T lymphocytes will be practicable in various fields associated with the treatment of autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus, anticancer vaccines, cell therapy products, and other diseases.
[Technical Solution]
[0010] An aspect of the present invention provides a culture composition for inducing activated T lymphocytes from peripheral blood.
[0011] Another aspect of the present invention provides a culture kit for inducing activated T lymphocytes from peripheral blood. [0012] Another aspect of the present invention provides a method for inducing activated T lymphocytes using the culture composition and the culture kit.
[0013] According to an aspect of the present invention, there is provided a culture composition for inducing activated T lymphocytes from peripheral blood, comprising adenosine triphosphate (ATP) , a divalent cation, a chloride, vitamin B, insulin, Granulocyte colony-stimulating factors (G-CSF) , erythropoietin, and a solvent.
[0014] The induced T cells may be CD3 and CD25 cell marker positive .
[0015] The divalent cation may be one or more selected from the group consisting of Zn2+, Cu2+, Mg2+, and Cr2+.
[0016] The chloride may be one or more selected from the group consisting of potassium chloride and bromine chloride.
[0017] The vitamin B may be vitamin B6.
[0018] The solvent may be distilled water.
[0019] The culture composition may comprise 10-15 mg of adenosine triphosphate (ATP) , 5-7 mg of the divalent cation, 80-100 mg of the chloride, 45-55 mg of vitamin B, 3-7 I.U. of insulin, 5-15 g of Granulocyte colony-stimulating factors (G-CSF), 5-15 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc .
[0020] According to another aspect of the present invention, there is provided a culture kit for inducing activated T lymphocytes from peripheral blood, comprising an airtight sterile container, which contains the above culture composition, and in which internal pressure is controlled to be equal to external pressure thereof.
[0021] According to another aspect of the present invention, there is provided a method for inducing activated T lymphocytes from peripheral blood, 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 culture composition, and in which internal pressure is controlled to be equal to external pressure thereof; and inserting the buffy coat and plasma poured container into a low temperature incubator and incubating the buffy coat and plasma.
[0022 ] The incubation may be carried out for a period of f om about 50 hours to about 200 hours.
[0023 ] The incubation may be carried out at a temperature from about 37 to 39 °C.
[0024] The induced T cells may be CD3 and CD25 cell marker positive .
[Advantageous Effects]
[0025] With the culture composition, the culture kit of the present invention for inducing activated T lymphocytes from peripheral blood, and the method for inducing activated T lymphocytes using the same, a large amount of activated T lymphocytes can easily be obtained from peripheral blood obtained from adults. Thus, the obtained activated T lymphocytes can be used in various fields associated with the treatment of autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus, and in cell therapy products which can induce the reduction and destruction of tumors, and the like. [Description of Drawings]
[0026] 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:
FIG. 1 is a diagram illustrating the differentiation process from hematopoietic stem cells to mature blood cells;
FIG. 2(a) illustrates the culture composition of the present invention, FIG. 2(b) illustrates the result directly after pouring the buffy coat and plasma into the culture composition of the present invention, and FIG. 2(c) illustrates the result after incubating the culture kit of the present invention for 4 weeks; and
FIGS. 3 to 5 illustrate results obtained by incubating peripheral blood samples for 28 days under the same conditions using the culture kit of the present invention, into which three groups of peripheral blood samples were poured, immunohistochemistry staining was carried out, and then peripheral blood samples were observed under a microscope (X400) . In respective FIGS. 3 to 5, (a) illustrates the result of immunohistochemistry staining for CD3, and (b) illustrates the result of immunohistochemistry staining for CD25.
[Best Mode]
[0027] Exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
[0028] According to the present invention, there is provided a culture composition for inducing activated T lymphocytes from peripheral blood. According to the culture composition of the present invention, T cells can be obtained from differentiation-completed blood corpuscles in peripheral blood, in particular, leukocytes, by induction.
[0029] According to the present invention, activated T lymphocytes which are CD3 and CD25 positive can be obtained from leukocytes obtained from peripheral blood. CD3 is a cell marker which is commonly expressed in all T lymphocytes and proves that unknown cells are T cells, and among these T cells, CD25 -expressing T cells are considered to be activated T lymphocytes.
[0030] More particularly, according to the present invention, activated T lymphocytes can be induced from a buffy coat isolated from peripheral blood, and especially, activated T lymphocytes can be induced from leukocytes contained in the buffy coat.
[0031] The culture composition 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.
[0032] In the present invention, water may be used for the solvent, and preferably, distilled water may be used for the solvent.
[0033] Adenosine triphosphate is also known as ATP, and functions as an energy source for facilitating and maintaining the differentiation into activated T lymphocytes in the culture composition.
[0034] The ATP may be comprised in an amount of 10 to 15 mg, and preferably, 11 to 13 mg, based on the final volume of the culture composition of the present invention being 8 cc. Where the ATP is comprised in an amount of less than 10 mg, there may be a problem in which the directed incubation into activated T lymphocytes may not be carried out sufficiently, and where the ATP is comprised in an amount of more than 15 mg, there may be a problem in which the blood corpuscles may differentiate into a type of cell other than activated T lymphocytes .
[0035] The divalent cation is a component designed to stabilize cell membrane and balance electrolytes in the culture composition of the present invention, and may be one or more selected from the group consisting of Zn+, Cu2+, g2+, and Cr2+, and preferably, Zn+. 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.
[0036] 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.
[0037 ] The divalent cation may be comprised in an amount of 5 to 7 mg, and preferably, 5.2 to 6.6 mg, based on the final volume of the culture composition of the present invention being 8 cc.
[0038] Where the divalent cation is comprised in an amount of less than 5 mg, there may be a problem in which the directed differentiation into activated T lymphocytes may be insufficient. Where the divalent cation is comprised in an amount of more than 7 mg, there is a concern that cells may die due to high osmotic pressure in a culture medium.
[0039] The chloride comprised in the present invention plays a role in stabilizing cations and maintaining a suitable concentration of a culture medium in the culture composition. The chloride may¬ be one or more selected from the group consisting of potassium chloride and bromine chloride, and preferably, potassium chloride .
[0040] The chloride may be comprised in an amount of 100 to 200 mg, and preferably, 130 to 170 mg, based on the final volume of the culture composition of the present invention being 8 cc.
[0041] 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 is raised too high. When the osmotic pressure is lowered or raised by too much as above, it may have adverse affects on a cell culture, and the induction of activated T lymphocytes may not be achieved smoothly.
[0042] The vitamin B may be vitamin B6 (pyridoxine) , and the vitamin B acts as a catalyst for cell division in the culture composition.
[0043] 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 culture composition of the present invention being 8 cc.
[0044] Where vitamin B6 is comprised in an amount of less than 45 mg, there is a problem with the efficiency of cell division and differentiation, and where vitamin B6 is comprised in an amount of more than 55 mg, there is a concern for an occurrence of cytotoxicity.
[0045] Insulin acts as a messenger for cell differentiation in the culture composition, and R (Regular) insulin may be used for the insulin. [0046] The insulin may be comprised in an amount of 3 to 7 I.U., and preferably, 4 to 6 I.U. , based on the final volume of the culture composition of the present invention being 8 cc.
[0047] Where the insulin is comprised in an amount of less than 3 I.U., there may be a problem in which the efficiency of cell division and differentiation may be deteriorated, and where the insulin is comprised in an amount of more than 7 I.U., there is a possibility of an occurrence of cell overproliferation and apoptosis.
[0048] Granulocyte colony-stimulating factors (G-CSF) , also known as filgrastim, act as a messenger for inducing differentiation in the cell differentiation process, and recombinant G-CSF may be used for the G-CSF.
[0049] The G-CSF may be comprised in an amount of 5 to 15 \ig, and preferably, 8 to 12 μς, based on the final volume of the culture composition of the present invention being 8 cc.
[0050] Where the G-CSF is comprised in an amount of less than 5 g, there may be a problem in which the efficiency of cell division and differentiation may be deteriorated, and where the G-CSF is comprised in an amount of more than 15 pg, there is concern for an occurrence of cell overproliferation and apoptosis.
[0051] The erythropoietin also plays a role in inducing differentiation in the cell differentiation process, and recombinant erythropoietin may be used for the erythropoietin.
[0052] The erythropoietin may be comprised in an amount of 5 to 15 I.U., and preferably, 8 to 12 I.U., based on the final volume of the culture composition of the present invention being 8 cc. [0053] Where the erythropoietin is comprised in an amount of less than 5 I .U. , there may be a problem in which the efficiency of cell division and differentiation may be deteriorated, and where the erythropoietin is comprised in an amount of more than 15 I .U. , there is a concern for an occurrence of cell overproliferation and apoptosis .
[0054] Most preferably, the culture composition of the present invention may comprise 10-15 mg of adenosine triphosphate (ATP) , 5-7 mg of the divalent cation, 80-100 mg of the chloride, 45-55 mg of vitamin B, 3-7 I.U. of insulin, 5-15 μς of Granulocyte colony-stimulating factors (G-CSF) , 5-15 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc.
[0055] Meanwhile, according to the present invention, there is provided a culture kit for inducing activated T lymphocytes from peripheral blood. The culture kit of the present invention comprises an airtight container containing the above described culture composition of the present invention.
[0056] Furthermore, the culture kit may comprise an airtight sterile container, in which internal pressure is controlled to be equal to external pressure thereof .
[0057] A container usable for the culture kit of the present invention may be a sterilized airtight container. Materials for the container are not particularly limited, and any container suitable for culture growth 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 culture composition and allow cells to be cultured, may be used. Most preferably, vials may be used for the container.
[0058] The container may contain the culture composition of the present invention in an amount from about 7/50 to about 9/50 of the overall volume.
[0059] Furthermore, according to the present invention, there is provided a method for inducing activated T lymphocytes from peripheral blood.
[0060] The method of the present invention uses the above culture composition and the culture kit of the present invention, and more particularly, comprises mixing peripheral 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 culture composition of the present invention, and in which internal pressure is controlled to be equal to external pressure thereof; and inserting the buffy coat and plasma poured container into a low temperature incubator and incubating the buffy coat and plasma.
[0061] The culture composition and the culture kit comprising the same, which are used for the method for inducing activated T lymphocytes from peripheral blood of the present invention, are the same as described above.
[0062] More particularly, for the method for inducing activated T lymphocytes 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. [0063 ] Here, the blood may be peripheral blood, which is 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 .
[0064 ] Here, the usable anticoagulant may be, but not limited to, one or more selected from the group of sodium citrate and heparin.
[0065] 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.
[0066] 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 .
[0067] 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.
[0068] 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 xbuffy coat' . The topmost layer occupies about 54%, and is comprised of plasma.
[0069] Into the airtight sterile container, which contains the culture composition of the present invention, and in which 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 syringe needle into a rubber stopper (rubber packing) , followed by removing the syringe needle if the pressures are in equilibrium but is not limited thereto.
[0070] Subsequently, the buffy coat layer and the plasma layer- poured container are incubated in a low temperature incubator. Here, the low temperature incubation may be carried out for 50 to 200 hours, and preferably, for 70 to 160 hours . Where the incubation is carried out for less than 50 hours, there may be a problem in which the incubation may be insufficient. Where the incubation is carried out for more than 200 hours, there may be a problem in which cell concentration may increase excessively in the culture composition and thus, apoptosis or cell suicide may occur.
[0071] The incubation may be carried out at a temperature from 37 to 39 °C, and more preferably, at about 38 °C. Where the incubation is carried out at a temperature less than 37 °C, there may be a problem in which the blood corpuscles may differentiate into a type of cell other than activated T lymphocytes. Where the incubation is carried out at more than 39 °C, there is a concern of an occurrence of cell damage from heat.
[0072] 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]
[0073] Embodiments
[0074] <Embodiments>
[0075] 1. Preparation of culture kit comprising culture medium
[0076] 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.
[0077] 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 culture kit, an airtight 50 cc glass bottle.
[0078] For the buffy coat and plasma obtained by the centrifugation of 30 cc of blood, the culture composition comprising the following components described in Table 1 was contained in the culture kit, and distilled water was added as a solvent thereto to make up the final volume of the whole composition to 8 cc.
[0079] [ Table l]
Figure imgf000016_0001
Figure imgf000017_0001
[0080] 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 .
[0081] FIG. 2(a) illustrates the culture composition of the present invention, FIG. 2(b) illustrates the result directly after pouring the buffy coat and plasma into the culture composition of the present invention, and FIG. 2(c) illustrates the result after incubating the culture kit of the present invention for 4 weeks.
[0082] 2. Induction process of activated T lymphocytes from peripheral blood
[0083] Blood samples were collected from three volunteers. Three groups of the culture kits as prepared in "1." were formed and incubated for 28 days under the same conditions. The incubation temperature was 38 °C.
[0084] 3. Confirmation of results for T cell induction
[0085] (1) Immunohistochemistry staining [0086] After incubation, each sample contained in each culture kit 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. Bond-MAX (Leica) equipment was used in immunohistochemistry staining.
[0087] 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. 3, FIG. 4, and FIG. 5, respectively.
[0088] A list of antibodies in the immunofluorescent experiment for confirming the cell differentiation into activated T lymphocytes is CD3 and CD25. Markers for immunohistochemistry staining used in this experiment were as per the following Table 2.
[0089] [ Table l]
Figure imgf000018_0001
[0090] Meanwhile, the information on the staining reagent for immunohistochemistry used in this experiment is as follows:
Leica Microsystems Bond TM Polymer Refine Detection
Catalog No: DS9800
[0091] In FIGS. 3 to 5, (a) illustrates the result of immunohistochemistry staining for CD3, and (b) illustrates the result of immunohistochemistry staining for CD25.
[0092] (2) Conclusion
[0093] Referring to FIGS. 3 to 5, as shown in the above experiment, after 4 weeks of incubation, the lymphoid cell marker CD3 was expressed and CD25 was also expressed. This indicates that activated T lymphocytes are obtained by the present invention. Experiment for three groups showed the same results .
[0094] Referring to FIGS. 3 to 5, as the result of the experiment, cell components having two groups of nuclei comprising a small amount of lymphocyte-like nuclei with a large amount of platelets and a small amount of cytoplasms, and a small amount of lymphocyte or monocyte-like nuclei with a large amount of cytoplasms were identified. Both groups of cells exhibited strong immunoreactivity for both CD3 and CD25.
[0095] That is, this result confirms that activated T lymphocytes were induced' from mature peripheral leukocytes with the use of the culture composition, the culture kit, and the method of the present invention.
[0096] 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 culture composition for inducing activated T lymphocytes from peripheral blood, 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 culture composition of claim 1, wherein the induced activated T cells are CD3 and CD25 cell marker positive.
[Claim 3]
The culture composition of claim 1, wherein the divalent cation is one or more selected from the group consisting of Zn2+, Cu2+, Mg2+, and Cr2+.
[Claim 4]
The culture composition of claim 1, wherein the chloride is one or more selected from the group consisting of potassium chloride and bromine chloride.
[Claim 5]
The culture composition of claim 1, wherein the vitamin B is vitamin B6 (pyridoxine) .
[Claim 6]
The culture composition of claim 1, wherein the solvent is distilled water. [Claim 7]
The culture composition of claim 1, comprising 10-15 mg of adenosine triphosphate (ATP) , 5-7 mg of the divalent cation, 80-100 mg of the chloride, 45-55 mg of vitamin B, 3-7 I.U. of insulin, 5-15 μς of Granulocyte colony-stimulating factors (G-CSF) , 5-15 I.U. of erythropoietin, and the solvent as a remainder such that a final overall volume is 8 cc.
[Claim 8]
A culture kit for inducing activated T lymphocytes from peripheral blood, comprising an airtight sterile container, which contains the culture composition 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 inducing activated T lymphocytes from peripheral blood, 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 culture composition of any of claims 1 to 7, and in which internal pressure is controlled to be equal to external pressure thereof; and
inserting the buffy coat and plasma poured container into a low temperature incubator and incubating the buffy coat and plasma. [Claim 10]
The method of claim 9, wherein the incubation is carried out for a period of from about 50 hours to about 200 hours.
[Claim ll]
The method of claim 9, wherein the incubation is carried out at from about 37 to about 39 °C.
[Claim 12]
The method of claim 9, wherein the induced activated T cells are CD3 and CD25 cell marker positive.
PCT/KR2012/006318 2012-07-06 2012-08-08 Culture composition for inducing activated t lymphocytes from peripheral blood, culture kit comprising the same, and method for inducing activated t lymphocytes from peripheral blood using the same WO2014007421A1 (en)

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