WO2006011682A1 - リンパ球の保存及び輸送方法 - Google Patents
リンパ球の保存及び輸送方法 Download PDFInfo
- Publication number
- WO2006011682A1 WO2006011682A1 PCT/JP2005/014365 JP2005014365W WO2006011682A1 WO 2006011682 A1 WO2006011682 A1 WO 2006011682A1 JP 2005014365 W JP2005014365 W JP 2005014365W WO 2006011682 A1 WO2006011682 A1 WO 2006011682A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lymphocytes
- transported
- mononuclear cells
- peripheral blood
- lymphocyte
- Prior art date
Links
- 210000004698 lymphocyte Anatomy 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000004321 preservation Methods 0.000 claims abstract 2
- 210000004369 blood Anatomy 0.000 claims description 24
- 239000008280 blood Substances 0.000 claims description 24
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 20
- 206010028980 Neoplasm Diseases 0.000 claims description 8
- 201000011510 cancer Diseases 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 claims description 5
- 108010002350 Interleukin-2 Proteins 0.000 claims description 4
- 238000001802 infusion Methods 0.000 claims description 4
- 208000035473 Communicable disease Diseases 0.000 claims 2
- 210000005087 mononuclear cell Anatomy 0.000 claims 2
- 210000005259 peripheral blood Anatomy 0.000 claims 2
- 239000011886 peripheral blood Substances 0.000 claims 2
- 238000000926 separation method Methods 0.000 claims 2
- 210000004027 cell Anatomy 0.000 abstract description 32
- 238000003306 harvesting Methods 0.000 abstract description 3
- 239000011232 storage material Substances 0.000 abstract 1
- 238000002659 cell therapy Methods 0.000 description 8
- 210000002865 immune cell Anatomy 0.000 description 8
- 230000002062 proliferating effect Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 238000012136 culture method Methods 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000000644 isotonic solution Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229940105132 myristate Drugs 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- 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
- A61P35/00—Antineoplastic agents
-
- 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
- the present invention relates to a method for storing lymphocytes.
- the present invention also relates to a method for transporting lymphocytes.
- cancer malignant neoplasm
- immune cell therapy refers to collecting blood from a patient, separating and culturing lymphocytes contained in the blood, activating and / or proliferating the lymphocytes, activating and / or proliferating the lymphocytes. This refers to the treatment of returning activated lymphocytes (including activated and / or proliferated lymphocytes including lymphokine activated killer cells (hereinafter referred to as LAK cells)) to the patient's body.
- LAK cells lymphokine activated killer cells
- lymphocytes collected from the patient and the activated and Z or proliferated lymphocytes for a certain period of time in advance. There is a case.
- red blood cells or platelets used for transfusion In general, however, only the storage temperature of whole blood, red blood cells or platelets used for transfusion is known. Disclosure of the invention
- the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a technique for maintaining the lymphocyte viable cell rate and the IFN-a producing cell rate during storage and transportation.
- the viable cell ratio is maintained even after 10 hours or more, especially 24 hours or more.
- the rate of IFN-a producing cells can be kept high.
- the temperature 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.
- FIG. 1 is a graph showing changes in the percentage of viable cells over time when lymphocytes (L AK cells) are suspended in physiological saline and stored at 0, 6, 14 and 25 X.
- FIG. 2 is a graph showing the change over time in the rate of IFN—r producing cells when lymphocytes (L AK cells) are suspended in physiological saline and stored in 0, 6, 14 and 2.
- lymphocytes are collected and separated from the blood of patients with cancer and Z or infection.
- peripheral blood mononuclear cells may be separated after collection.
- any method for separating nucleated cells from red blood cells can be used as a method for separating peripheral blood mononuclear cells.
- Ficoll pack Ficol 1-Paque density gradient method is generally used.
- peripheral blood mononuclear cells By isolating peripheral blood mononuclear cells, the storage temperature range described above is expanded to 5 to 2 2 X.
- 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 lymphocyte refers to a T lymphocyte, a B lymphocyte, an NK cell, or an NKT cell.
- the culture method is not particularly limited, and any culture method generally used in the art may be used. 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 lymphocytes in a culture vessel such as a flask on which the anti-CD3 antibody is immobilized.
- the concentration of IL-2 is preferably added to the medium so as to have a concentration of 100 to 200 IUZmL.
- Culture 3 4 to 3 8 ° C, preferably at 3 7 ° C,. 2 to 1 0% conducted preferably at C_ ⁇ 2 under 5% culture period from one day to 2 days 0, especially 1 ⁇ 2 weeks is preferred.
- the medium that can be used is not particularly limited, but AIM-V medium (Invitrogen), RPM 1-1640 medium (Invitrogen), Dulbecco's modified medium (Invitrogen), Iskov medium (Invitrogen), KBM Commercially available media used for cell culture such as media (Kohjin Bio) and AL y S media (Cell Science Laboratory) can be used. If necessary, 5 to 20% of bovine serum, fetal bovine serum, human serum, human plasma, etc. can be added.
- the culture vessel is not particularly limited, and culture plates, petri dishes, flasks, bags, etc. that are usually used in the field can be used.
- the concentration for seeding each cell group can be freely set according to the situation.
- the lymphocytes separated and cultured from the blood thus collected and then harvested are prepared as an injection using a commonly used carrier.
- the carrier to be used is not particularly limited.
- an isotonic solution such as physiological saline, P B S (phosphate buffered physiological saline) and the like can be mentioned.
- 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 collected blood is stored and Z or transported at 16 to 22 ° C., so that peripheral blood mononuclear cells are well separated from the collected blood and proliferative. Can be maintained in a state.
- the storage temperature range is expanded to 5 to 22 while maintaining a high proliferative state.
- the lymphocytes obtained by culturing are stored and transported or transported at 0-6 ° C. Thereby, it is possible to obtain lymphocytes which 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 handling on the sample blood before culturing and the lymphocyte obtained after culturing, and further, by combining these handling, a more excellent effect can be obtained it can.
- culture method used here is not limited to the above-described conditions, but can also apply conditions commonly known in lymphocyte culture.
- Human peripheral blood mononuclear cells are seeded in anti-CD3 antibody (Orthoclone: Janssen Pharma) solid-phase flask (Sumitomo Beichikrite) and IL-2 is adjusted to 280 I UZmL in the medium (KBM400: Kojin Bio) And cultured (37 ° C, C 0 2 : 5%).
- lymphocytes After culturing for 14 days, lymphocytes (LAK cells) were collected by centrifugation and washed.
- the cultured lymphocytes were stored in physiological saline (0.2% human serum albumin) at a density of 5 ⁇ 10 7 ce 11 s / mL.
- the viable cell ratio after each storage time was determined by the PI (Propidiurn Iodide) method, and I FN-a producing cells.
- the rate was measured by the measurement method of intracellular site force by PMA (Phorbo 1 1 2 -Myristate 13-Ac etate) and ionomycin (I ono myc in) stimulation.
- Figure 1 shows the change in the viable cell ratio after 6, 30, 36, and 50 hours after harvesting at each storage temperature.
- Figure 2 shows the change in the IFN-producing cell rate after 6, 24, 30, 36, and 50 hours after harvesting at each storage temperature.
- the method of the present invention provides a method for storage and transport while maintaining a high lymphocyte viable cell rate and IFN-producing cell rate, It has excellent effects as a storage and Z or transport method for injections containing spheres.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006527902A JPWO2006011682A1 (ja) | 2004-07-30 | 2005-07-29 | リンパ球の保存及び輸送方法 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004-247556 | 2004-07-30 | ||
JP2004247556 | 2004-07-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2006011682A1 true WO2006011682A1 (ja) | 2006-02-02 |
Family
ID=35786406
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2005/014365 WO2006011682A1 (ja) | 2004-07-30 | 2005-07-29 | リンパ球の保存及び輸送方法 |
Country Status (2)
Country | Link |
---|---|
JP (2) | JPWO2006011682A1 (ja) |
WO (1) | WO2006011682A1 (ja) |
-
2005
- 2005-07-29 JP JP2006527902A patent/JPWO2006011682A1/ja active Pending
- 2005-07-29 WO PCT/JP2005/014365 patent/WO2006011682A1/ja active Application Filing
-
2011
- 2011-04-04 JP JP2011082603A patent/JP2011139711A/ja active Pending
Non-Patent Citations (7)
Also Published As
Publication number | Publication date |
---|---|
JP2011139711A (ja) | 2011-07-21 |
JPWO2006011682A1 (ja) | 2008-05-01 |
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