WO2006011681A1 - Procédé de conservation et procédé de transport pour du sang destiné à l'incubation de leucocytes, procédé de conservation et procédé de transport pour des monocytes du sang périphérique et procédé d'incubation de leu - Google Patents

Procédé de conservation et procédé de transport pour du sang destiné à l'incubation de leucocytes, procédé de conservation et procédé de transport pour des monocytes du sang périphérique et procédé d'incubation de leu Download PDF

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Publication number
WO2006011681A1
WO2006011681A1 PCT/JP2005/014364 JP2005014364W WO2006011681A1 WO 2006011681 A1 WO2006011681 A1 WO 2006011681A1 JP 2005014364 W JP2005014364 W JP 2005014364W WO 2006011681 A1 WO2006011681 A1 WO 2006011681A1
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WIPO (PCT)
Prior art keywords
blood
culture
leukocyte culture
peripheral blood
leukocyte
Prior art date
Application number
PCT/JP2005/014364
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English (en)
Japanese (ja)
Inventor
Hidemasa Jinguji
Atsutaka Noguchi
Shiho Sato
Kazutoshi Sato
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Medinet Co., Ltd
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Publication date
Application filed by Medinet Co., Ltd filed Critical Medinet Co., Ltd
Priority to JP2006527901A priority Critical patent/JPWO2006011681A1/ja
Publication of WO2006011681A1 publication Critical patent/WO2006011681A1/fr

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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
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0087—Purging against subsets of blood cells, e.g. purging alloreactive T cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • Leukocyte culture blood storage method Leukocyte culture blood storage method, transport method, peripheral blood mononuclear cell storage method, transport method and leukocyte culture method using them Technical Field
  • the present invention relates to a method for preserving blood for leukocyte culture, a method for transporting blood for leukocyte culture, a method for preserving peripheral blood mononuclear cells, and a method for transporting peripheral blood mononuclear cells. Furthermore, the present invention relates to a method for culturing leukocytes using them.
  • cancer malignant neoplasm
  • immune cell therapy refers to collecting blood from a patient, separating and culturing leukocytes contained in the blood, activating and Z or proliferating the leukocytes, and activating and / or proliferating the leukocytes.
  • This refers to a therapy in which leukocytes (including those prepared by using activated and / or expanded leukocytes as an instillation) are returned to the patient's body.
  • leukocytes including those prepared by using activated and / or expanded leukocytes as an instillation
  • activated autolymphocyte therapy including activated lymphocytes including L AK cells (Lymphophocine Acivated K i 1 1 er cells)
  • dendritic cell vaccines include CTL therapy (Cytoto Xic TL ymphocyte) Therapy).
  • the present invention has been made in view of the above circumstances, and provides a method for easily separating leukocytes from blood collected from a patient and storing and transporting or transporting them while maintaining high proliferation ability.
  • the purpose is to provide. Furthermore, it aims at providing the culture method using the said white blood cell.
  • the present invention by storing the blood for leukocyte culture at 16 to 22 ° C., it is possible to separate peripheral blood mononuclear cells after blood collection for 10 hours or more, especially 30 hours or more. It is possible to maintain a high proliferation ability.
  • it may be maintained with a normal thermostatic device, and when transported, it may be transported in a state of being placed in the thermostatic device.
  • the cold storage for maintaining the temperature of the present invention may be stored and Z or transported in a normally used cold storage box having excellent heat insulation.
  • blood collected from a patient may be subjected to an operation such as centrifugation in advance before storage to separate peripheral blood mononuclear cells.
  • Leukocytes in the present invention mean granulocytes such as eosinophils, basophils, and neutrophils; monocytes; lymphocytes such as T lymphocytes, B lymphocytes, and natural killer cells.
  • the leukocytes in the blood for culturing leukocytes preserved in this state maintain excellent separation and proliferation ability, including lymphocytes in immune cell therapy such as activated autolymphocyte therapy, dendritic cell vaccine, and CTL therapy. It is useful for the culture of leukocytes.
  • Figure 1 (a) shows the effect on the number of recovered cells at storage temperatures of 2 ° C, 6 ° C, 10 ° C, 14 ° C, 18 ° C, and 25, and Figure 1 (b) shows peripheral blood. Indicates the number of red blood cells mixed in the mononuclear cell fraction.
  • Figure 2 shows the effect on cell growth rates at 6 ° C, 10 ° C, 14 ° C, 18 ° C, and 25 ° C at storage temperature 2.
  • the vertical axis shows the magnification when the number of cells before culture is 1.
  • Figure 3 shows the effect on the number of recovered cells at storage temperatures of 16 and 18 ° (: 20 ° C, 22 ° C).
  • Figure 4 shows the effect on cell growth rate at storage temperatures of 16, 18 ° C, 20 ° C, and 22 ° C.
  • the vertical axis shows the magnification when the number of cells before culture is 1.
  • Figure 5 shows a storage temperature of 5 ° C for samples from which peripheral blood mononuclear cells were separated before storage. The number of recovered cells at 10 ° C, 15 ° C and 20 ° C is shown.
  • Figure 6 shows the cell growth rate at storage temperatures of 5 ° C, 10 ° C, 15 and 20 ° C for samples subjected to peripheral blood mononuclear cell separation before storage.
  • the vertical axis shows the magnification when the number of cells before culture is 1.
  • the collection method is not particularly limited, but a commonly used blood collection tube may be used.
  • peripheral blood mononuclear cells may be separated.
  • any method for separating nucleated cells from erythrocytes can be used as a method for separating peripheral blood mononuclear cells.
  • a method using a Ficoll pack (Ficol 1 -Paque) density gradient is generally used.
  • peripheral blood mononuclear cells By separating peripheral blood mononuclear cells, the storage temperature range described above is expanded to 5 to 22 ° C.
  • the isolated peripheral blood mononuclear cells may be suspended in any carrier as long as they are isotonic with cells, but are preferably suspended in autologous plasma.
  • lymphocytes are cultured.
  • the culture method is not particularly limited, and any culture method that is generally used in the field may be used, and when used for activated autolymphocyte therapy, a method using an anti-CD3 antibody and IL-12 is particularly preferable.
  • the anti-CD3 antibody may be added to the medium or immobilized on the culture vessel, but it is preferable to seed the lymphocytes on a culture vessel such as a flask in which the anti-D3 antibody is immobilized. It is preferable to add IL-12 so that the concentration in the medium is 100 to 2000 IU / mL.
  • Culture 34 ⁇ 38 ⁇ preferably at 37, 2-10%, preferably carried out at 5% of C0 2 conditions, the incubation period is from 1 day to 20 days, it is not preferable particularly about 1-2 weeks.
  • the medium that can be used is not particularly limited, but AIM-V medium (Invitrogen), RPM 1-1640 medium (Invitrogen), Dulbecco's modified Eagle medium (Invitrogen), Iskov medium (Invitrogen), KBM medium (Kohjin Bio), Commercially available media used for cell culture such as ALyS medium (Laboratory for Cell Science) can be used. If necessary, 5-20% bovine serum, fetal bovine serum, human serum, human plasma, etc. can be added.
  • the culture vessel is not particularly limited, and is usually for culture used in the field. Plates, petri dishes, flasks, bags, etc. can be used. The concentration for seeding each cell group can be freely set according to the situation.
  • the lymphocytes harvested after separation and culture from the collected blood are prepared as injections using commonly used carriers.
  • the carrier to be used is not particularly limited. For example, when it is prepared as an instillation agent, an isotonic solution such as physiological saline or PBS (phosphate buffered physiological saline) can be used. A serum component such as albumin may be added to the isotonic solution.
  • lymphocytes preserved in this state maintain a high viable cell rate and an IFN-a-producing cell rate, and are useful as an injection for immune cell therapy.
  • it is useful as an injection for use in immune cell therapy for cancer and Z or infection.
  • the separation of peripheral blood mononuclear cells from the collected blood is good and the proliferation is high. Can be maintained in a state.
  • the storage temperature range is expanded to 5 to 22 ° C while maintaining a high proliferative state.
  • lymphocytes obtained by culturing are stored and transported or transported at 0-6 ° C. This makes it possible to obtain lymphocytes that maintain a high viable cell rate and an IFN-producing cell rate while maintaining a high proliferative state. Therefore, in lymphocyte culture, there is an excellent effect by imposing different treatments on the sample blood before the culture and the lymphocytes obtained after the culture, and a more excellent effect can be obtained by combining these treatments.
  • the culture method used here may be cultured not only under the above-mentioned conditions but also under conditions generally known in lymphocyte culture.
  • lymphocyte LAK cell culture As described above, the case of lymphocyte LAK cell culture has been described in detail. However, other leukocytes may be cultured by a method generally known in the art. Example
  • vacutina CPT Blood was collected by Kuton (Dickinson).
  • the blood that entered the vacuum blood collection tube was stored at each temperature for 30 hours.
  • the number of collected cells was measured using a flow cytometer (Cytomics FC500: Beckman Cole Yuichi).
  • IL-2 is 280 I UZmL added to a cultured (37 ° C, C_ ⁇ 2: 5%) was performed.
  • the number of LAK cells after 7 days of culture was measured with a hemocytometer.
  • Figure la shows the number of T cells collected in peripheral blood mononuclear cell fractions from peripheral blood stored at 2, 6 ° C, 10 ° C, 14, 18 and 25 ° C for 30 hours.
  • b shows the number of red blood cells mixed in the peripheral blood mononuclear cell fraction.
  • Figure 2 shows LAK cells grown from peripheral blood mononuclear cells collected from peripheral blood stored at storage temperatures of 2 ° C, 6 ° C, 10 ° C, 14 ° C, 18 ° C, and 25 ° C for 30 hours. The growth rate was shown.
  • the storage temperature of LAK cells was 50 times at 18 ° C, 34 times at 14 ° C, and 20 times at others. When inducing LAK cells using stored blood, it was found that a storage temperature of around 18 is suitable.
  • Fig. 3 shows the number of T cells recovered from the peripheral blood stored for 30 hours at storage temperatures of 16 ° C, 18 ° C, 20, and 22 in the peripheral blood mononuclear cell fraction.
  • Figure 4 shows the proliferation rate of LA cells proliferated from peripheral blood mononuclear cells collected from peripheral blood stored at storage temperatures of 16 ° C, 18 ° C, 20 and 22 ° C for 30 hours.
  • 7.5 ml of peripheral blood was collected from a healthy donor into a vacuum blood collection tube (Baccutina CPT: Becton 'Dickinson').
  • Peripheral blood mononuclear cells were stored in autologous plasma for 30 hours at each temperature. The number of collected cells was measured using a flow cytometer (Cytomics FC 500: Beckman Cole).
  • IL-2 is 280 IU / was added to a mL culture (37 ° C, C0 2: 5%) was.
  • LAK cells The number of cells (LAK cells) after 8 days of culture was measured with a hemocytometer.
  • Figure 5 shows the number of sputum cells recovered from samples that were isolated from peripheral blood mononuclear cells before storage and stored at storage temperatures of 5 ° C, 10 ° C, 15 ° C, and 2 ° C for 30 hours.
  • Fig. 6 shows the proliferation rate of LAK cells grown from samples that were isolated from peripheral blood mononuclear cells before storage and stored at storage temperatures of 5, 10 ° C, 15, and 20 ° C for 30 hours.
  • LAK cell proliferation in samples from which peripheral blood mononuclear cells were isolated before storage was found to be 5 to 20 ° C, and the dependence on storage temperature was low.
  • the method of the present invention provides a method for easily separating leukocytes from blood and preserving and / or transporting them while maintaining high proliferation ability. It has an excellent effect as a method for storing and / or transporting blood for lymphocyte culture in therapy. In addition, high proliferation can be expected by culturing leukocytes isolated from these lymphocyte culture blood.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medical Preparation Storing Or Oral Administration Devices (AREA)

Abstract

L'invention concerne un procédé par lequel on peut facilement séparer les leucocytes du sang recueilli chez un patient et les conserver et/ou les transporter tout en conservant une aptitude à proliférer élevée, et ainsi on parvient à réaliser d'excellents procédés de conservation et/ou de transport pour le sang destiné à l'incubation de leucocytes devant être utilisé en thérapie cellulaire immunologique. L'invention concerne également un procédé d'incubation utilisant les leucocytes conservés et/ou transportés par les procédés ci-dessus. A savoir qu'on conserve le sang destiné à l'incubation de leucocytes à une température de 16 à 22°C de façon à pouvoir bien séparer les monocytes du sang périphérique 10 heures, en particulier 30 heures, après la collecte du sang et à pouvoir conserver une aptitude à proliférer élevée. Dans le cas où on sépare les monocytes du sang périphérique avant la conservation, la plage des températures convenant pour la conservation et/ou le transport va de 5 à 22°C.
PCT/JP2005/014364 2004-07-30 2005-07-29 Procédé de conservation et procédé de transport pour du sang destiné à l'incubation de leucocytes, procédé de conservation et procédé de transport pour des monocytes du sang périphérique et procédé d'incubation de leu WO2006011681A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006527901A JPWO2006011681A1 (ja) 2004-07-30 2005-07-29 白血球培養用血液の保存方法、輸送方法、末梢血単核球の保存方法、輸送方法及びそれらを用いた白血球の培養方法

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JP2004-247555 2004-07-30
JP2004247555 2004-07-30

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010505408A (ja) * 2006-10-06 2010-02-25 オックスフォード イミュノテック リミテッド 調製方法
RU2495568C1 (ru) * 2012-05-23 2013-10-20 Общество с ограниченной ответственностью "ФАРМКОНСАЛТИНГ" (ООО "ФАРМКОНСАЛТИНГ") Способ хранения донорской эритроцитарной массы

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] MAJSKY A. ET AL: "Enhancement of the specific cytotoxic reactivity of HLA antigens on lymphocytes stored at +4 degrees C for 24 hours", XP002999302, Database accession no. 79087014 *
KIKUCHI T. ET AL: "Ko-CD3 Kotai o Mochiite Yudo Shita Lymphokine Activated Killer (LAK) Saibo no Koshuyosho Koka Narabini Rinpa Kyu Hyomen Marker no Kento", NOSHINKEI, vol. 42, no. 6, 1990, pages 575 - 580, XP002999301 *
MAJSKY A. ET AL: "Enhancement of the specific cytotoxic reactivity of HLA antigens on lymphocytes stored at +4 degrees C for 24 hours", FOLIA HAEMATOL.INT.MAG.KLIN.MORPHOL.BLUTFORSCH., vol. 105, no. 4, 1978, pages 506 - 508 *
MULLER-STEINHARDT M. ET AL: "Impact of storage at 22 degrees C and citrate anticoagulation on the cytokine secretion of mononuclear leukocytes", VOX SANG, vol. 75, no. 1, 1998, pages 12 - 17, XP002998499 *
NAGAHATA H. ET AL: "Effects of Ficoll Concentration and Blood Storage on the Separation of Lymphocytes From Bovine Peripheral Blood", J.COLL.DAIRYING, vol. 9, 1982, pages 391 - 399, XP002998498 *
SON B.K. ET AL: "Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight", CLIN.DIAGN.LAB.IMMUNOL., vol. 3, no. 3, 1996, pages 260 - 264, XP002998500 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010505408A (ja) * 2006-10-06 2010-02-25 オックスフォード イミュノテック リミテッド 調製方法
US9090871B2 (en) 2006-10-06 2015-07-28 Oxford Immunotec Limited Cell-mediated immunoassays
RU2495568C1 (ru) * 2012-05-23 2013-10-20 Общество с ограниченной ответственностью "ФАРМКОНСАЛТИНГ" (ООО "ФАРМКОНСАЛТИНГ") Способ хранения донорской эритроцитарной массы

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