CN106701827A - Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) - Google Patents
Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) Download PDFInfo
- Publication number
- CN106701827A CN106701827A CN201611102044.0A CN201611102044A CN106701827A CN 106701827 A CN106701827 A CN 106701827A CN 201611102044 A CN201611102044 A CN 201611102044A CN 106701827 A CN106701827 A CN 106701827A
- Authority
- CN
- China
- Prior art keywords
- cells
- cell
- cryopreservation
- antibody genes
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 57
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 title claims abstract description 33
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 title claims abstract description 33
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 title claims abstract description 32
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 26
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title abstract description 9
- 238000004321 preservation Methods 0.000 title description 2
- 230000009466 transformation Effects 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 108010083359 Antigen Receptors Proteins 0.000 claims abstract description 13
- 102000006306 Antigen Receptors Human genes 0.000 claims abstract description 13
- 230000001131 transforming effect Effects 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 241000713666 Lentivirus Species 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 112
- 238000005138 cryopreservation Methods 0.000 claims description 24
- 210000002966 serum Anatomy 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 19
- 102000000588 Interleukin-2 Human genes 0.000 claims description 18
- 108010002350 Interleukin-2 Proteins 0.000 claims description 18
- 238000007710 freezing Methods 0.000 claims description 18
- 230000008014 freezing Effects 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 17
- 239000008188 pellet Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 230000006044 T cell activation Effects 0.000 claims description 7
- 239000007825 activation reagent Substances 0.000 claims description 7
- 238000012546 transfer Methods 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000684 flow cytometry Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000004083 survival effect Effects 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 239000003792 electrolyte Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 241000700605 Viruses Species 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 241000204031 Mycoplasma Species 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000007774 longterm Effects 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000013190 sterility testing Methods 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000011287 therapeutic dose Methods 0.000 claims description 3
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 238000004113 cell culture Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000003053 toxin Substances 0.000 claims description 2
- 231100000765 toxin Toxicity 0.000 claims description 2
- 239000012679 serum free medium Substances 0.000 claims 1
- 238000009169 immunotherapy Methods 0.000 abstract description 8
- 230000003211 malignant effect Effects 0.000 abstract description 7
- 208000004736 B-Cell Leukemia Diseases 0.000 abstract description 4
- 239000000427 antigen Substances 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 208000028018 Lymphocytic leukaemia Diseases 0.000 abstract description 3
- 208000003747 lymphoid leukemia Diseases 0.000 abstract description 3
- 230000008685 targeting Effects 0.000 abstract description 3
- 239000006143 cell culture medium Substances 0.000 description 5
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012360 testing method Methods 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/14—Mechanical aspects of preservation; Apparatus or containers therefor
- A01N1/142—Apparatus
- A01N1/144—Apparatus for temperature control, e.g. refrigerators or freeze-drying apparatus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70517—CD8
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Hematology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种表达CD19和CD20抗体基因崁合抗原受体T细胞的转化及扩增培养与保存方法，该方法首先利用重组慢病毒将人T细胞转化；然后将转化后的人T细胞进行扩增培养；将所获得的表达CD19和CD20抗体基因嵌合抗原受体的人T细胞进行冰冻保存。本发明通过构建靶向CD19和CD20抗体基因嵌合抗原受体慢病毒，并将其高效率的转染至T淋巴细胞，对转染后的淋巴T细胞进行了扩增培养并保存，使CD19和CD20作为恶性淋巴细胞白血病免疫治疗的一种有效的靶抗原，对于恶性B淋巴细胞白血病细胞免疫治疗具有重要临床意义。
The invention discloses a method for transforming, amplifying, culturing and preserving T cells expressing CD19 and CD20 antibody genes combined with antigen receptors. In the method, human T cells are first transformed by recombinant lentivirus; and then transformed human T cells are transformed. Carry out expansion culture; cryopreserve the obtained human T cells expressing CD19 and CD20 antibody gene chimeric antigen receptors. In the present invention, by constructing a chimeric antigen receptor lentivirus targeting CD19 and CD20 antibody genes, and transfecting it to T lymphocytes with high efficiency, the transfected lymphoid T cells are amplified, cultured and preserved, so that CD19 As an effective target antigen for immunotherapy of malignant lymphocytic leukemia, CD20 has important clinical significance for immunotherapy of malignant B lymphocytic leukemia.
Description
技术领域：Technical field:
本发明属于细胞工程技术领域，涉及一种用嵌合抗原受体修饰后T细胞的扩增培养与保存方法，具体涉及一种表达CD19和CD20抗体基因崁合抗原受体T细胞的转化及扩增培养与保存方法。The invention belongs to the technical field of cell engineering, and relates to a method for expanding, cultivating and preserving T cells modified with chimeric antigen receptors, in particular to a method for transforming and expanding T cells expressing CD19 and CD20 antibody genes combined with antigen receptors Growth and storage methods.
背景技术：Background technique:
淋巴T细胞是构成细胞免疫的主要成分，而利用基因工程使T细胞表达嵌合抗原受体能使T细胞转化成为具有特异性杀伤肿瘤细胞的效应细胞，是一种有前景的肿瘤免疫治疗策略。嵌合性抗原受体应用的关键是确定一种肿瘤相关抗原，这种抗原具有在肿瘤细胞表面过表达或高表达，而在正常组织无表达或低表达，CD19在早期B细胞的发育中扮演重要的角色，所有的急性白血病都表达CD19。CD20表达于90％以上的B淋巴瘤细胞和正常B淋巴细胞，而在造血干细胞、原始B淋巴细胞、正常血细胞以及其他组织上不表达。因此，CD19和CD20可作为恶性淋巴细胞白血病免疫治疗的一种有效的靶抗原。构建靶向CD19和CD20抗体基因嵌合抗原受体慢病毒，并将其高效率的转染至T淋巴细胞，对转染后的淋巴T细胞扩增培养并保存是对恶性B淋巴细胞白血病细胞免疫治疗的前提关键。因此，本发明提供一种用于人T细胞的慢病毒转化及扩增培养与保存的方法，对于恶性B淋巴细胞白血病细胞免疫治疗具有重要临床意义。Lymphatic T cells are the main components of cellular immunity, and using genetic engineering to make T cells express chimeric antigen receptors can transform T cells into effector cells that can specifically kill tumor cells, which is a promising strategy for tumor immunotherapy . The key to the application of chimeric antigen receptors is to determine a tumor-associated antigen, which is overexpressed or highly expressed on the surface of tumor cells, but has no or low expression in normal tissues. CD19 plays a role in the development of early B cells. important role, all acute leukemias express CD19. CD20 is expressed in more than 90% of B lymphoma cells and normal B lymphocytes, but not expressed in hematopoietic stem cells, primitive B lymphocytes, normal blood cells and other tissues. Therefore, CD19 and CD20 can be used as an effective target antigen for immunotherapy of malignant lymphocytic leukemia. Construct chimeric antigen receptor lentiviruses targeting CD19 and CD20 antibody genes, and transfect them to T lymphocytes with high efficiency. The transfected lymphoid T cells are expanded, cultured and preserved for malignant B lymphocytic leukemia cells The premise of immunotherapy is critical. Therefore, the present invention provides a method for lentiviral transformation, expansion, culture and preservation of human T cells, which has important clinical significance for the immunotherapy of malignant B lymphocytic leukemia cells.
发明内容：Invention content:
针对现有技术存在的问题，本发明的目的旨在提供一种表达CD19和CD20抗体基因崁合抗原受体T细胞的转化及扩增培养与保存方法。Aiming at the problems existing in the prior art, the object of the present invention is to provide a method for transforming, expanding, culturing and preserving T cells expressing CD19 and CD20 antibody genes combined with antigen receptors.
为达到上述目的，本发明采取以下技术方案：To achieve the above object, the present invention takes the following technical solutions:
表达CD19和CD20抗体基因崁合抗原受体T细胞的转化及扩增培养与保存方法，包括如下步骤：A method for transforming, expanding, culturing and preserving T cells expressing CD19 and CD20 antibody genes combined with antigen receptors, comprising the following steps:
S1：将分离获得的CD4和CD8阳性T细胞悬浮于含人血清AB和细胞介素-2(IL-2)的培养基中，经T细胞激活试剂在37℃，5％CO2孵育箱中进行培养激活；S1: Suspend the isolated CD4 and CD8 positive T cells in the medium containing human serum AB and interleukin-2 (IL-2), and incubate at 37°C with 5% CO 2 after T cell activation reagents perform culture activation;
所述含有人血清AB和细胞介素-2的培养基为含2％的人血清AB和200单位/mL的细胞介素-2的T细胞培养基，其是一种无异源成分的无血清培养基，专门用于人T细胞的生长和扩增。The medium containing human serum AB and interleukin-2 is a T cell culture medium containing 2% human serum AB and 200 units/mL of interleukin-2, which is a non-xenogeneous component-free Serum medium specifically designed for the growth and expansion of human T cells.
所述T细胞激活试剂采用TransAct CD3/28。The T cell activation reagent uses TransAct CD3/28.
S2：经T细胞激活试剂激活后，将T细胞转移至离心管中，离心后取上清；S2: After being activated by the T cell activation reagent, the T cells are transferred to a centrifuge tube, and the supernatant is taken after centrifugation;
S3：将上述上清倾倒至另一离心管中备用，轻弹有细胞沉淀的离心管底部使细胞分散，将细胞重新悬浮于收集备用的上清中，移出少量细胞悬液计数；S3: Pour the above supernatant into another centrifuge tube for later use, lightly flick the bottom of the centrifuge tube with cell pellets to disperse the cells, resuspend the cells in the collected supernatant, remove a small amount of cell suspension and count;
S4：用备用的离心上清调整细胞浓度，然后接种于培养板中；S4: Adjust the cell concentration with the spare centrifuged supernatant, and then inoculate in the culture plate;
所述备用的离心上清调整细胞浓度为0.5X106/mL，按0.5X106/1mL/孔的量接种于六孔培养板中。The spare centrifuged supernatant was adjusted to a cell concentration of 0.5X10 6 /mL, and seeded in a six-well culture plate at an amount of 0.5X10 6 /1 mL/well.
S5：将慢病毒从-80℃条件下取出，于室温中融化后，加入到上述含有T细胞的培养板中，混匀，置37℃，5％CO2孵育箱中进行孵育；S5: Take out the lentivirus from -80°C, melt it at room temperature, add it to the above-mentioned culture plate containing T cells, mix well, and incubate in a 37°C, 5% CO 2 incubator;
所述加入含有T细胞的培养板中的慢病毒用量根据病毒的感染滴度，按照感染复数(multiplicity ofinfection，MOI)5计算病毒用量。The amount of lentivirus added to the culture plate containing T cells is calculated according to the infection titer of the virus, and the virus amount is calculated according to the multiplicity of infection (MOI) 5.
S6：待孵育结束时，向培养板中加入含人血清AB和细胞介素-2的培养基，混匀，置37℃，5％CO2孵育箱中进行孵育(如果收集备用的离心上清没有用完，可与上述培养基混合然后加入孔中)；S6: At the end of the incubation, add the culture medium containing human serum AB and interleukin-2 to the culture plate, mix well, and incubate in a 37°C, 5% CO 2 incubator (if the centrifuged supernatant is collected If not used up, it can be mixed with the above medium and added to the well);
所述含有人血清AB和细胞介素-2的培养基为含2％的人血清AB和200单位/mL的细胞介素-2的T细胞培养基，其是一种无异源成分的无血清培养基，专门用于人T细胞的生长和扩增。The medium containing human serum AB and interleukin-2 is a T cell culture medium containing 2% human serum AB and 200 units/mL of interleukin-2, which is a non-xenogeneous component-free Serum medium specifically designed for the growth and expansion of human T cells.
S7：孵育后进行T细胞的收集，并取少量细胞计数计算总细胞数和细胞存活率，同时用流式细胞仪检测CD19和CD20的表达水平；S7: Collect T cells after incubation, and count a small number of cells to calculate the total cell number and cell survival rate, and use flow cytometry to detect the expression levels of CD19 and CD20;
S8：将收集的T细胞转移至加有含人血清AB和细胞介素-2的培养基的G-Rex100培养装置中，置37℃，5％CO2孵育箱中进行扩增培养，中间无需换液或其他处理，尽量减少对细胞的干扰(根据培养板的细胞采用G-Rex100培养装置的数量，如一个六孔板的细胞用一个G-Rex100)；S8: Transfer the collected T cells to the G-Rex100 culture device added with the medium containing human serum AB and interleukin-2, and place them in a 37°C, 5% CO 2 incubator for expansion culture, no need in the middle Change the medium or other treatments to minimize the interference to the cells (according to the number of G-Rex100 culture devices used for the cells in the culture plate, such as one G-Rex100 for cells in a six-well plate);
所述含有人血清AB和细胞介素-2的培养基为含2％的人血清AB和200单位/mL的细胞介素-2的T细胞培养基，其是一种无异源成分的无血清培养基，专门用于人T细胞的生长和扩增。The medium containing human serum AB and interleukin-2 is a T cell culture medium containing 2% human serum AB and 200 units/mL of interleukin-2, which is a non-xenogeneous component-free Serum medium specifically designed for the growth and expansion of human T cells.
S9：待细胞培养结束后，收获扩增细胞，并取少量细胞计数计算总细胞数和细胞存活率，同时用流式细胞仪检测CD19和CD20的表达水平，另取部分细胞进行支原体检测和内毒素检测；S9: After the cell culture is over, the expanded cells are harvested, and a small number of cells are counted to calculate the total cell number and cell survival rate. At the same time, the expression levels of CD19 and CD20 are detected by flow cytometry, and some cells are taken for mycoplasma detection and internal control. Toxin detection;
S10：将剩余细胞进行离心，并弃上清，再用复方电解质注射液洗涤细胞一次，再次进行离心后，即得细胞沉淀悬浮细胞；S10: Centrifuge the remaining cells, discard the supernatant, wash the cells once with compound electrolyte injection, and centrifuge again to obtain cell pellets and suspension cells;
S11：按照不同的治疗剂量将细胞沉淀悬浮细胞置于细胞冻存液中，并转移到细胞冻存袋中，此时移出1mL细胞进行无菌检测；S11: Place the cell pellet and suspend cells in the cell cryopreservation solution according to different therapeutic doses, and transfer them to the cell cryopreservation bag. At this time, remove 1 mL of cells for sterility testing;
所述细胞冻存液包括24％的复方电解质注射液，10％的DMSO，8％的人血清白蛋白。The cell cryopreservation solution includes 24% compound electrolyte injection, 10% DMSO, and 8% human serum albumin.
S12：在将细胞沉淀悬浮细胞于细胞冻存液之前，启动细胞冻存程序；S12: Before suspending the cell pellet in the cell freezing solution, start the cell freezing procedure;
所述细胞冻存程序，具体包括如下步骤：The cell cryopreservation program specifically includes the following steps:
a.启动细胞冻存程序，使细胞冻存室温度降至4℃；a. Start the cell cryopreservation program and lower the temperature of the cell cryopreservation chamber to 4°C;
b.将含有细胞的冻存袋移至冻存室，继续运行冻存程序；b. Move the cryopreservation bag containing the cells to the cryopreservation room and continue the cryopreservation procedure;
c.细胞中温度降低1℃/min至-11.5℃；c. Decrease the temperature in the cell by 1°C/min to -11.5°C;
d.冻存室中温度降低20℃/min至-55℃；d. The temperature in the freezing chamber is lowered by 20°C/min to -55°C;
e.冻存室中温度降低15℃/min至-25℃；e. The temperature in the freezing chamber is lowered by 15°C/min to -25°C;
f.冻存室中温度降低1℃/min至-45℃；f. The temperature in the freezing chamber is lowered by 1°C/min to -45°C;
g.冻存室中温度降低10℃/min至-100℃。g. Decrease the temperature in the freezing chamber by 10°C/min to -100°C.
S13：待细胞温度降到-90℃，细胞冻存完成；S13: When the temperature of the cells drops to -90°C, the cryopreservation of the cells is completed;
S14：将冻存好的细胞移至液氮中进行长期保存。S14: Move the frozen cells to liquid nitrogen for long-term storage.
本发明表达CD19和CD20抗体基因崁合抗原受体T细胞的转化及扩增培养与保存方法，通过构建靶向CD19和CD20抗体基因嵌合抗原受体慢病毒，并将其高效率的转染至T淋巴细胞，对转染后的淋巴T细胞进行了扩增培养并保存，使CD19和CD20作为恶性淋巴细胞白血病免疫治疗的一种有效的靶抗原，对于恶性B淋巴细胞白血病细胞免疫治疗具有重要临床意义。The method for transforming, amplifying, culturing and preserving T cells expressing CD19 and CD20 antibody genes and antigen receptors in the present invention is to construct a chimeric antigen receptor lentivirus targeting CD19 and CD20 antibody genes, and transfect them with high efficiency To T lymphocytes, the transfected lymphoid T cells are expanded, cultured and preserved, so that CD19 and CD20 can be used as an effective target antigen for the immunotherapy of malignant lymphocytic leukemia, and have the potential for immunotherapy of malignant B lymphocytic leukemia. important clinical significance.
附图说明：Description of drawings:
图1是本发明实施例中表达CD19和CD20抗体基因嵌合抗原受体T细胞的扩增结果示意图；Fig. 1 is a schematic diagram of the expansion results of chimeric antigen receptor T cells expressing CD19 and CD20 antibody genes in the embodiment of the present invention;
图2是实施例中表达CD19和CD20抗体基因嵌合抗原受体T细胞培养于G-Rex100培养装置中的图片；Fig. 2 is a picture of chimeric antigen receptor T cells expressing CD19 and CD20 antibody genes cultured in the G-Rex100 culture device in the embodiment;
图3是实施例中冰冻保存的表达CD19和CD20抗体基因嵌合抗原受体T细胞。Fig. 3 is the cryopreserved chimeric antigen receptor T cells expressing CD19 and CD20 antibody genes in the embodiment.
具体实施方式：detailed description:
下面对本发明的技术方案作进一步的说明，但并不局限于此，凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的保护范围中。The technical solution of the present invention will be further described below, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered by the protection scope of the present invention middle.
实施例1Example 1
一种表达CD19和CD20抗体基因崁合抗原受体T细胞的转化及扩增培养与保存方法，包括如下步骤：A method for transforming, expanding, culturing and preserving T cells expressing CD19 and CD20 antibody genes combined with antigen receptors, comprising the following steps:
S1：将分离获得的CD4和CD8阳性T细胞悬浮于含2％的人血清AB和200单位/mL的细胞介素-2的T细胞培养基中，经T细胞激活试剂在37℃，5％CO2孵育箱中进行培养激活；S1: Suspend the isolated CD4 and CD8 positive T cells in the T cell culture medium containing 2% human serum AB and 200 units/mL of interleukin-2, after T cell activation reagent at 37 °C, 5% Culture activation in a CO 2 incubator;
S2：经T细胞激活试剂激活作用一天后，将T细胞转移至离心管中，300r离心10min后取上清；S2: After being activated by the T cell activating reagent for one day, transfer the T cells to a centrifuge tube, centrifuge at 300r for 10min, and take the supernatant;
S3：将上清倾倒至另一离心管中备用，轻弹有细胞沉淀的离心管底部使细胞分散，将细胞重新悬浮于收集备用的上清中，移出少量细胞悬液计数；S3: Pour the supernatant into another centrifuge tube for later use, lightly flick the bottom of the centrifuge tube with cell pellets to disperse the cells, resuspend the cells in the collected supernatant, remove a small amount of cell suspension and count;
S4：用备用的离心上清调整细胞浓度为0.5X106/mL，然后按0.5X106/1mL/孔的量接种于六孔培养板中；S4: Use the spare centrifuged supernatant to adjust the cell concentration to 0.5X10 6 /mL, and then inoculate in a six-well culture plate at an amount of 0.5X10 6 /1 mL/well;
S5：将慢病毒从-80℃条件下取出，于室温中融化后，加入到上述含有T细胞的六孔培养板中，混匀，置37℃，5％CO2孵育箱中进行孵育6h；S5: Take out the lentivirus from -80°C, melt it at room temperature, add it to the above-mentioned six-well culture plate containing T cells, mix well, and incubate in a 37°C, 5% CO 2 incubator for 6 hours;
S6：待孵育结束时，向六孔培养板中每孔加入1mL含2％的人血清AB和200单位/mL的细胞介素-2的T细胞培养基，混匀，置37℃，5％CO2孵育箱中进行孵育4天(如果收集备用的离心上清没有用完，可与上述培养基混合然后加入孔中)；S6: At the end of the incubation, add 1 mL of T cell culture medium containing 2% human serum AB and 200 units/mL of interleukin-2 to each well of the six-well culture plate, mix well, and place at 37°C, 5% Incubate in a CO 2 incubator for 4 days (if the collected centrifuged supernatant is not used up, it can be mixed with the above medium and added to the well);
S7：孵育4天后进行T细胞的收集，并取少量细胞计数计算总细胞数和细胞存活率，同时用流式细胞仪检测CD19和CD20的表达水平；S7: After 4 days of incubation, T cells were collected, and a small amount of cells were counted to calculate the total cell number and cell survival rate, and the expression levels of CD19 and CD20 were detected by flow cytometry;
S8：将收集的T细胞转移至加有含2％的人血清AB和200单位/mL的细胞介素-2的T细胞培养基的G-Rex100培养装置中，如图2所示，至培养基体积到450mL，置37℃，5％CO2孵育箱中进行扩增培养8天，中间无需换液或其他处理，尽量减少对细胞的干扰；S8: Transfer the collected T cells to the G-Rex100 culture device added with T cell medium containing 2% human serum AB and 200 units/mL of interleukin-2, as shown in Figure 2, until cultured Base volume to 450mL, place in 37°C, 5% CO 2 incubator for expansion culture for 8 days, without changing medium or other treatment in the middle, to minimize interference to cells;
S9：待细胞培养至7天结束后，收获扩增细胞，并取少量细胞计数计算总细胞数和细胞存活率，同时用流式细胞仪检测CD19和CD20的表达水平，另取部分细胞进行支原体检测和内毒素检测；S9: After the cells have been cultured for 7 days, the expanded cells are harvested, and a small number of cells are counted to calculate the total cell number and cell survival rate. At the same time, the expression levels of CD19 and CD20 are detected by flow cytometry, and some cells are collected for mycoplasma detection and endotoxin testing;
S10：将剩余细胞进行300g离心10min，并弃上清，再用复方电解质注射液洗涤细胞一次，再次进行300g离心10min后，即得细胞沉淀悬浮细胞；S10: Centrifuge the remaining cells at 300g for 10 minutes, discard the supernatant, wash the cells once with compound electrolyte injection, and centrifuge again at 300g for 10 minutes to obtain the cell pellet and suspension cells;
S11：如图3所示，按照不同的治疗剂量将细胞沉淀悬浮细胞置于细胞冻存液中，并转移到细胞冻存袋中，此时移出1mL细胞进行无菌检测；S11: As shown in Figure 3, place the cell pellet and suspend cells in the cell cryopreservation solution according to different therapeutic doses, and transfer them to the cell cryopreservation bag. At this time, remove 1 mL of cells for sterility testing;
S12：在将细胞沉淀悬浮细胞于细胞冻存液之前，启动细胞冻存程序；S12: Before suspending the cell pellet in the cell freezing solution, start the cell freezing procedure;
所述细胞冻存程序，具体包括如下步骤：The cell cryopreservation program specifically includes the following steps:
a.启动细胞冻存程序，使细胞冻存室温度降至4℃；a. Start the cell cryopreservation program and lower the temperature of the cell cryopreservation chamber to 4°C;
b.将含有细胞的冻存袋移至冻存室，继续运行冻存程序；b. Move the cryopreservation bag containing the cells to the cryopreservation room and continue the cryopreservation procedure;
c.细胞中温度降低1℃/min至-11.5℃；c. Decrease the temperature in the cell by 1°C/min to -11.5°C;
d.冻存室中温度降低20℃/min至-55℃；d. The temperature in the freezing chamber is lowered by 20°C/min to -55°C;
e.冻存室中温度降低15℃/min至-25℃；e. The temperature in the freezing chamber is lowered by 15°C/min to -25°C;
f.冻存室中温度降低1℃/min至-45℃；f. The temperature in the freezing chamber is lowered by 1°C/min to -45°C;
g.冻存室中温度降低10℃/min至-100℃。g. Decrease the temperature in the freezing chamber by 10°C/min to -100°C.
S13：待细胞温度降到-90℃，细胞冻存完成；S13: When the temperature of the cells drops to -90°C, the cryopreservation of the cells is completed;
S14：将冻存好的细胞移至液氮中进行长期保存。S14: Move the frozen cells to liquid nitrogen for long-term storage.
如图1所示，为表达CD19和CD20抗体基因嵌合抗原受体T细胞的扩增结果可知：T细胞培养第5天至第13天之间细胞扩增的数量增至1750*106到2200*106，然后可计算总细胞数和细胞存活率。As shown in Figure 1, the results of the expansion of chimeric antigen receptor T cells expressing CD19 and CD20 antibody genes show that the number of expanded cells increased to 1750*10 6 to 1750*106 to 2200*10 6 , then the total cell number and cell viability can be calculated.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611102044.0A CN106701827A (en) | 2016-12-05 | 2016-12-05 | Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611102044.0A CN106701827A (en) | 2016-12-05 | 2016-12-05 | Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701827A true CN106701827A (en) | 2017-05-24 |
Family
ID=58935638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611102044.0A Pending CN106701827A (en) | 2016-12-05 | 2016-12-05 | Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701827A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107970258A (en) * | 2017-11-20 | 2018-05-01 | 英普乐孚生物技术(上海)有限公司 | A kind of Chimeric antigen receptor T cell preparation |
| CN111479613A (en) * | 2017-10-18 | 2020-07-31 | 凯德药业股份有限公司 | Methods of administering chimeric antigen receptor immunotherapy |
| CN111978394A (en) * | 2020-07-13 | 2020-11-24 | 珠海龄值生物科技有限公司 | Preparation method of polyclonal preparation for COVID-19 patient specific immunotherapy |
| CN113957046A (en) * | 2020-07-20 | 2022-01-21 | 东莞市东阳光生物药研发有限公司 | Culture medium for T cell lentivirus infection and application |
| CN114615886A (en) * | 2019-08-29 | 2022-06-10 | 得克萨斯大学体系董事会 | Cell cryopreservation culture medium |
| US11793834B2 (en) | 2018-12-12 | 2023-10-24 | Kite Pharma, Inc. | Chimeric antigen and T cell receptors and methods of use |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105384825A (en) * | 2015-08-11 | 2016-03-09 | 南京传奇生物科技有限公司 | Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof |
| CN105980402A (en) * | 2013-12-20 | 2016-09-28 | 弗雷德哈钦森癌症研究中心 | Tagged chimeric effector molecules and receptors thereof |
| CN106163547A (en) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | Use Chimeric antigen receptor treatment cancer |
-
2016
- 2016-12-05 CN CN201611102044.0A patent/CN106701827A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105980402A (en) * | 2013-12-20 | 2016-09-28 | 弗雷德哈钦森癌症研究中心 | Tagged chimeric effector molecules and receptors thereof |
| CN106163547A (en) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | Use Chimeric antigen receptor treatment cancer |
| CN105384825A (en) * | 2015-08-11 | 2016-03-09 | 南京传奇生物科技有限公司 | Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111479613A (en) * | 2017-10-18 | 2020-07-31 | 凯德药业股份有限公司 | Methods of administering chimeric antigen receptor immunotherapy |
| CN107970258A (en) * | 2017-11-20 | 2018-05-01 | 英普乐孚生物技术(上海)有限公司 | A kind of Chimeric antigen receptor T cell preparation |
| CN107970258B (en) * | 2017-11-20 | 2020-11-10 | 英普乐孚生物技术(上海)有限公司 | Chimeric antigen receptor T cell preparation |
| US11793834B2 (en) | 2018-12-12 | 2023-10-24 | Kite Pharma, Inc. | Chimeric antigen and T cell receptors and methods of use |
| CN114615886A (en) * | 2019-08-29 | 2022-06-10 | 得克萨斯大学体系董事会 | Cell cryopreservation culture medium |
| CN111978394A (en) * | 2020-07-13 | 2020-11-24 | 珠海龄值生物科技有限公司 | Preparation method of polyclonal preparation for COVID-19 patient specific immunotherapy |
| CN111978394B (en) * | 2020-07-13 | 2021-05-11 | 珠海龄值生物科技有限公司 | Preparation method of polyclonal preparation for COVID-19 patient specific immunotherapy |
| CN113957046A (en) * | 2020-07-20 | 2022-01-21 | 东莞市东阳光生物药研发有限公司 | Culture medium for T cell lentivirus infection and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106701827A (en) | Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) | |
| CN104938477B (en) | A kind of CIK cell frozen stock solution and cryopreservation methods | |
| CN102994449A (en) | Method for in-vitro amplification of NK cells | |
| CN107904204A (en) | A kind of preparation method of NK cells | |
| CN105994254A (en) | Cryopreservation solution and cryopreservation method of DC cell | |
| CN114085812B (en) | Mesenchymal stem cell population with high expression of CD106 and/or CD142 and reduced expression, and preparation method and application thereof | |
| CN103374548A (en) | High-efficiency amplification culture solution for tumor infiltration lymphocytes | |
| CN105543169A (en) | Preparation method and application of human TSCMs (T memory stem cells) | |
| Solari et al. | Human dendritic cells and transplant outcome | |
| CN107574148A (en) | A kind of NK (NK cells) culture medium and preparation method thereof | |
| CN105505871A (en) | Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells | |
| CN101914497B (en) | Clinical N-CIK cell culture and quality control and identification kit and application | |
| CN103800897A (en) | Preparation method and kit for dendritic cell vaccine loaded by tumor specific antigenic epitope polypeptide | |
| Caserta et al. | IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells | |
| CN106399241A (en) | Medium for gamma delta T cells amplified in vitro and method | |
| CN117187179A (en) | Method for culturing CIK cells by utilizing cryopreserved umbilical cord blood mononuclear cells | |
| CN105219711A (en) | The culture system of a kind of CIKs cell and DC-CIKs cell | |
| CN103834614A (en) | Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof | |
| US20230295566A1 (en) | Method for producing tumor-infiltrating t-lymphocytes (til) and their use as cellular therapeutics for the treatment of human tumors | |
| CN105132445A (en) | Receptor protein, T lymphocyte and NK cells for specifically identifying cancer cells | |
| CN103571913B (en) | Method for detecting killing activity of human (gamma)(delta)T cells against K562 cell strain | |
| WO2024067673A1 (en) | Identification, in vitro amplification, and application method of memory cd8 t cells specific for tumor antigen | |
| CN105462925A (en) | Preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by PD-1 antibody | |
| CN106479973A (en) | A kind of external IAK immunocyte cultural method | |
| CN117551607A (en) | Culture method of malignant hydrothorax/ascites TIL cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |
|
| RJ01 | Rejection of invention patent application after publication |