CN113832101A - 自然杀伤细胞体外高效扩增的制备方法 - Google Patents
自然杀伤细胞体外高效扩增的制备方法 Download PDFInfo
- Publication number
- CN113832101A CN113832101A CN202111030087.3A CN202111030087A CN113832101A CN 113832101 A CN113832101 A CN 113832101A CN 202111030087 A CN202111030087 A CN 202111030087A CN 113832101 A CN113832101 A CN 113832101A
- Authority
- CN
- China
- Prior art keywords
- cells
- culture
- cell
- natural killer
- interleukin
- 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.)
- Withdrawn
Links
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 41
- 230000003321 amplification Effects 0.000 title claims abstract description 15
- 238000000338 in vitro Methods 0.000 title claims abstract description 15
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 62
- 102000003812 Interleukin-15 Human genes 0.000 claims description 12
- 108090000172 Interleukin-15 Proteins 0.000 claims description 12
- 102000000588 Interleukin-2 Human genes 0.000 claims description 12
- 108010002350 Interleukin-2 Proteins 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 11
- 210000005087 mononuclear cell Anatomy 0.000 claims description 9
- 210000005259 peripheral blood Anatomy 0.000 claims description 9
- 239000011886 peripheral blood Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 102000013462 Interleukin-12 Human genes 0.000 claims description 5
- 108010065805 Interleukin-12 Proteins 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 230000000415 inactivating effect Effects 0.000 claims description 5
- 108010067306 Fibronectins Proteins 0.000 claims description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 4
- 229930182816 L-glutamine Natural products 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 210000004700 fetal blood Anatomy 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 210000000601 blood cell Anatomy 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 102000003814 Interleukin-10 Human genes 0.000 claims description 2
- 108090000174 Interleukin-10 Proteins 0.000 claims description 2
- 241001505901 Streptococcus sp. 'group A' Species 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 229940117681 interleukin-12 Drugs 0.000 claims description 2
- 102000016359 Fibronectins Human genes 0.000 claims 1
- 239000000243 solution Substances 0.000 description 13
- 230000002147 killing effect Effects 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 102100037362 Fibronectin Human genes 0.000 description 3
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 3
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 3
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 102000001398 Granzyme Human genes 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 210000002993 trophoblast Anatomy 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101100518501 Mus musculus Spp1 gene Proteins 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000004395 cytoplasmic granule Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000010013 cytotoxic mechanism Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000002978 thoracic duct Anatomy 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
- C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2312—Interleukin-12 (IL-12)
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2315—Interleukin-15 (IL-15)
-
- 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
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex
-
- 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
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及生物领域，具体提供了一种自然杀伤细胞体外高效扩增的制备方法，所述方法包括预处理培养瓶，接种低密度单个核细胞以及有效抗体和细胞因子等的添加，所述方法成本低，能够显著提高NK细胞的扩增数量和扩增纯度。
Description
技术领域
本发明属于生物技术领域，具体涉及一种在体外扩增自然杀伤细胞的制备方法。
背景技术
70年代初美国国家癌症研究院(National Cancer Institute,USA)NCI的研究人员在研究T细胞对靶细胞的特异杀伤时发现正常对照小鼠的脾细胞可以象免疫小鼠的脾细胞一样杀伤某些肿瘤细胞，继而发现正常人外周血淋巴细胞也可以天然杀伤某些癌细胞。这些细胞的杀伤功能不需要预先免疫或致敏，故取名为自然杀伤细胞((natural killercells)。NK细胞是与T、B细胞并列的第三类群淋巴细胞。NK细胞数量较少，在外周血中约占淋巴细胞总数的10％，在脾内约有3％～4％，也可出现在肺脏、肝脏和肠粘膜，但在胸腺、淋巴结和胸导管中罕见。
NK细胞较大，含有胞浆颗粒，故称大颗粒淋巴细胞。以CD56+、CD16+、CD3-为免疫细胞特征，是一种特殊的淋巴细胞。NK通过识别细胞表面抗原簇self-MHC来区分正常细胞和非正常细胞。面对于正常细胞，NK处于非激活、非攻击状态；对于非正常细胞，包括病原体、被病毒感染的细胞、突变细胞、癌变细胞，一旦识别，就开启细胞毒(cytotoxicity)方式展开攻击并最终破坏靶细胞。NK细胞的细胞毒原理基本有两种，一种是NK细胞释放穿孔蛋白(perforin)和颗粒酶(granzyme)从而破坏靶细胞的膜完整性，导致靶细胞裂解；另一种是NK细胞通过引发细胞凋亡路径来杀死靶细胞。NK细胞可非特异直接杀伤靶细胞，这种天然杀伤活性既不需要预先由抗原致敏，也不需要抗体参与，且无MHC限制。之所以称作“自然杀手”，因为NK细胞与人体其它的150多种白细胞都不同，它不需要接受免疫系统的特殊指令，也不需要其它细胞的配合，自己单独就能识别和攻击外来细胞(如癌细胞)和病毒，在医学上被称作是“人体抵抗癌细胞和病毒感染细胞的第一道防线”。
目前，NK细胞培养存在两种培养方式：1、滋养层细胞(人肿瘤细胞，即人白血病K562细胞)培养方式。首先获得具有不分裂不增殖但仍保持代谢活性的滋养层细胞。该细胞通过基因工程技术进行改造，再经过钴60辐照，细胞膜表面稳定表达多种细胞因子，在多种细胞因子协同作用下，外周血单个核细胞中的自然杀伤细胞得到定向激活和扩增。该种培养方式的价格相对便宜，仅有3000元左右。从培养结果来看，细胞收获量可达到20亿/L的水平，NK表型阳性率达到80％-90％左右。如Korean J Lab Med 2009；29:89-96，Koehl U etal.Klin
2005；217:345–350)；(Blood，15March 2008，Vol.111，No.6，pp.3155-3162)等2、纯因子细胞培养方式：顾名思义就是使用相应的细胞因子来刺激单个核细胞，向NK细胞方向诱导，并配合相应的细胞培养基，使NK细胞大量增殖。纯因子细胞培养技术以其安全性高而著称，其原因在于所使用的细胞因子，都是体内环境原本就存在的。培养过程相当于模拟体内环境，促进NK细胞的激活及大量增殖。从培养结果来看，细胞收获量可达到15-25亿/L的水平，NK表型CD3-CD56+达到40％-90％左右。如：FehnigerT A.ShahMH.TurnerM Jeta1.Differentialcytokine and chemokinegene expression by human NKcellsfollowingactiva-tion withIL-18orIL-15incombination withIL-12：implications for the innateimmuneresponse[J].JImmunol，1999；162：4511-4520；EspluguesE，Vega-RamosJ，CartoixaD eta1.Induction of tumor NK—cellimmunitybyanti-CD69 antibodytherapy[J].Blood，2005；105：43994406等,但由于NK细胞在体内数量较少，制约了其大规模研究试验。因此，研制高效的体外扩增技术是NK细胞过继免疫治疗研究进展的关键。
发明内容
本发明提供一种自然杀伤细胞体外高效扩增的制备方法，具体的，提供了由不同来源的血液细胞在体外快速、高效扩增NK细胞的方法。根据本发明所述方法包括采用至少一种生长因子和有效浓度、有效暴露时间和有效持续时间培养包含NK细胞的细胞群；而且接种低密度单个核细胞以及有效抗体和细胞因子等的添加，所述方法成本低，能够显著提高NK细胞的扩增数量和扩增纯度。
本发明自然杀伤细胞体外高效扩增的制备方法，其特征在于，包括以下步骤：
步骤一：从血液样品中分离得到的单个核细胞按照0.5～5×106个/ml细胞密度接种到预处理的培养瓶中，所接种的培养基含有10～50ng/ml A群链球菌OK432、10～50ug/ml抗CD3单克隆抗体、500～1000U/ml白介素-2、10～50ng/ml白介素-15、100～500ug/ml白介素-12、L-谷氨酰胺50～100mg/ml、和2～5％的自体灭活血浆或商品化的灭活血浆，5％CO237℃培养箱中培养3～5天；
步骤二：将细胞以0.5～1×105/ml半量更换培养液，加入到新的抗CD16单克隆抗体包被的培养瓶中，培养液加500～1000U/ml白介素-2、10～50ng/ml白介素-15，换培养袋继续培养，每2～4更换加量的培养液并加同浓度的IL-2和IL-15，同时2～5％的自体灭活血浆或商品化的病毒灭活血浆，培养至21天收获细胞。
进一步地，预处理的培养瓶的制备方法：使用包括100～600ng/ml抗CD16单克隆抗体、0.1～1ug/ml纤维黏连蛋白(Fn)的D-PBS包被，4℃避光过夜，或37℃避光至少2小时。
进一步地，自体灭活血浆的制备方法：将外周血移至离心管，2000～3000rpm，离心5～20min，吸取上清液；将上清液56℃，30min条件下灭活，然后4℃静置15min,2000～4000rpm离心，5～20min，再次吸取上清液制得，4℃保存。
进一步地，细胞来源是外周血、脐血、骨髓或任何人体血液细胞。本发明提供的一种在体外扩增自然杀伤细胞的制备方法，所述方法包括预处理培养瓶，接种低密度单个核细胞以及有效抗体和细胞因子等的添加，所述方法成本低，能够显著提高NK细胞的扩增数量和扩增纯度。
附图说明
图1是在第0/3/5/7/10/12/17/21天分别检测得到的NK细胞数。
脐带血NK细胞倍增曲线：细胞总数360.4亿/2L体系；CD3-CD56+：73.9％。
外周血NK细胞倍增曲线：细胞总数亿327.82/2L体系；CD3-CD56+：86.3％。
具体实施方式
实施例1：
本实施例提供的一种自然杀伤细胞体外高效扩增的制备方法的制备方法，所述方法包括
步骤一：将抗100ng/mlCD16单克隆抗体(浓度)、纤维黏连蛋白(Fn)0.1ug/ml的D-PBS包被4℃避光过夜，或37℃避光至少2小时，第二天用前洗涤2次，注意不要冲洗包被面。
步骤二：加PBMS细胞0.5X106/ml到X-vivo 15培养液中，加入抗50ug/mlCD3单克隆抗体，50ng/mlOK432，1000U/mlIL-2，50ng/mlIL-15、500ug/mlIL-12、L-谷氨酰胺100mg/ml、和5％的自体灭活血浆中，并放入步骤一预处理的瓶中，5％CO2 37℃培养箱中培养6天，然后1200rpm，离心10min，收获细胞。
步骤二所述的自体灭活血浆的制备：将外周血移至离心管，2000～3000rpm，离心5～20min，吸取上清液；将上清液56℃，30min条件下灭活，然后4℃静置15min，2000～4000rpm离心，5～20min，再次吸取上清液制得，4℃保存。
步骤三：将细胞以0.5X105/ml到培养液中，加入到新的100ng/mlCD16单克隆抗体(浓度)包被的培养瓶中，培养液加1000U/ml白介素-2、50ng/ml白介素-15，换培养袋继续培养，每2～4更换加量的培养液并加同浓度的IL-2和IL-15，同时2～5％的自体灭活血浆或商品化的病毒灭活血浆，培养至21天收获细胞；
实施例2：
步骤一：制备自体灭活血浆：采集的外周血2000r/min离心10min得到血浆，血浆56℃水浴45min灭活补体，然后4℃静置15min,灭活后再次离心4000r/min离心20min取上清液，4℃冷藏备用。
步骤二：使用600ng/ml抗CD16单克隆抗体、1ug/ml纤维黏连蛋白(Fn)的D-PBS包被，4℃避光过夜，或37℃避光至少2小时；
步骤三:离心沉淀细胞用生理盐水重悬稀释后采用Ficoll法分离得到单个核细胞，生理盐水洗涤离心3次。将得到的单个核细胞按照5×106/ml浓度，接种于预先包被液处理的培养瓶中，每瓶完全培养基加入GT-T551 H3培养液，加入抗10ug/mlCD3单克隆抗体，10ng/mlOK432，500U/mlIL-2，10ng/mlIL-15、100ug/mlIL-12、L-谷氨酰胺50mg/ml和5％的自体灭活血浆中，置于37℃、体积分数5％CO2培养箱中进行培养3天；
步骤四：第4天，将培养的细胞按1000rpm/min离心15min，弃上清，用新鲜的完全培养基悬浮细胞，并调整细胞密度为5×105/ml浓度，置于600ng/ml抗CD16单克隆抗体包被的培养瓶中,置于GT-T551 H3培养液中，培养液加500U/ml白介素-2、10ng/ml白介素-15，换培养袋继续培养，每2～4更换加量的培养液并加同浓度的IL-2和IL-15，同时2～5％的自体灭活血浆或商品化的灭活血浆，培养至21天收获细胞。
实施例3：NK细胞对K562细胞株杀伤活性检测:
取对数生长期K562细胞，离心换液后将细胞浓度调整为4×105/ml将细胞轻轻吹打混匀后，接种于96孔板中，每孔100μl细胞悬液。按照效靶比按10:1/20:1/30:1/40:1分别调整细胞浓度，接种于96孔板，每孔100μl。铺板后将96孔板置于37℃、5％CO2培养箱中孵育24h后，每孔加入20μl CCK8溶液，37℃孵育1.5h，利用酶标仪在450nm处检测吸光度(A)值。按照如下公式计算效应细胞的杀伤活性：杀伤活性(％)＝[靶细胞对照组A值－(实验组A值－效应细胞对照组A值)]/靶细胞对照组A值×100。靶细胞对照组为单纯K562细胞，效应细胞对照组为单纯脐血NK细胞和外周血NK细胞。将体外培养的效应细胞分别与靶细胞作用，随着效靶比的升高，杀伤活性增强。见表1：
Claims (4)
1.自然杀伤细胞体外高效扩增的制备方法，其特征在于，包括以下步骤：
步骤一：从血液样品中分离得到的单个核细胞按照0.5～5×106个/ml细胞密度接种到预处理的培养瓶中，所接种的培养基含有10～50ng/ml A群链球菌OK432、10～50ug/ml抗CD3单克隆抗体、500～1000U/ml白介素-2、10～50ng/ml白介素-15、100～500ug/ml白介素-12、L-谷氨酰胺50～100mg/ml、和2～5％的自体灭活血浆或商品化的灭活血浆，5％CO2 37℃培养箱中培养3～5天；
步骤二：将细胞以0.5～1×105/ml半量更换培养液，加入到新的抗CD16单克隆抗体包被的培养瓶中，培养液加500～1000U/ml白介素-2、10～50ng/ml白介素-15，换培养袋继续培养，每2～4更换加量的培养液并加同浓度的IL-2和IL-15，同时2～5％的自体灭活血浆或商品化的灭活血浆，培养至21天收获细胞。
2.根据权利要求1所述的自然杀伤细胞体外高效扩增的制备方法，其特征在于:预处理的培养瓶的制备方法：使用包括100～600ng/ml抗CD16单克隆抗体、0.1～1ug/ml纤维黏连蛋白(Fn)的D-PBS包被，4℃避光过夜，或37℃避光至少2小时。
3.根据权利要求1所述的自然杀伤细胞体外高效扩增的制备方法，其特征在于：自体灭活血浆的制备方法：将外周血移至离心管，2000～3000rpm，离心5～20min，吸取上清液；将上清液56℃，30min条件下灭活，然后4℃静置15min,2000～4000rpm离心，5～20min，再次吸取上清液制得，4℃保存。
4.根据权利要求1所述的自然杀伤细胞体外高效扩增的制备方法，其特征在于：细胞来源是外周血、脐血、骨髓或任何人体血液细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111030087.3A CN113832101A (zh) | 2021-09-03 | 2021-09-03 | 自然杀伤细胞体外高效扩增的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111030087.3A CN113832101A (zh) | 2021-09-03 | 2021-09-03 | 自然杀伤细胞体外高效扩增的制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113832101A true CN113832101A (zh) | 2021-12-24 |
Family
ID=78962144
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111030087.3A Withdrawn CN113832101A (zh) | 2021-09-03 | 2021-09-03 | 自然杀伤细胞体外高效扩增的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113832101A (zh) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102112600A (zh) * | 2008-07-29 | 2011-06-29 | 株式会社绿十字 | 自然杀伤细胞的增殖方法 |
| KR20170000798A (ko) * | 2015-06-24 | 2017-01-03 | (주)차바이오텍 | 자연살해세포의 증식 방법 및 자연살해세포 증식용 조성물 |
| CN107326008A (zh) * | 2017-08-09 | 2017-11-07 | 上海莱馥医疗科技有限公司 | 一种从外周血中高效高纯度扩增自然杀伤细胞的方法 |
| CN107475196A (zh) * | 2017-10-09 | 2017-12-15 | 天津长和生物技术有限公司 | 自然杀伤细胞培养基质及自然杀伤细胞的扩增培养方法 |
| CN109666640A (zh) * | 2019-01-14 | 2019-04-23 | 武汉睿健医药科技有限公司 | 自然杀伤细胞体外纯培养的方法 |
| CN110511907A (zh) * | 2019-05-16 | 2019-11-29 | 安徽瑞达健康产业有限公司 | 一种体外稳定扩增高纯度、高细胞毒活性nk细胞的方法 |
| CN111690610A (zh) * | 2020-07-22 | 2020-09-22 | 暨赛再生医学科技有限公司 | 一种高效诱导培养制备自然杀伤性nk细胞的方法 |
-
2021
- 2021-09-03 CN CN202111030087.3A patent/CN113832101A/zh not_active Withdrawn
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102112600A (zh) * | 2008-07-29 | 2011-06-29 | 株式会社绿十字 | 自然杀伤细胞的增殖方法 |
| KR20170000798A (ko) * | 2015-06-24 | 2017-01-03 | (주)차바이오텍 | 자연살해세포의 증식 방법 및 자연살해세포 증식용 조성물 |
| CN107326008A (zh) * | 2017-08-09 | 2017-11-07 | 上海莱馥医疗科技有限公司 | 一种从外周血中高效高纯度扩增自然杀伤细胞的方法 |
| CN107475196A (zh) * | 2017-10-09 | 2017-12-15 | 天津长和生物技术有限公司 | 自然杀伤细胞培养基质及自然杀伤细胞的扩增培养方法 |
| CN109666640A (zh) * | 2019-01-14 | 2019-04-23 | 武汉睿健医药科技有限公司 | 自然杀伤细胞体外纯培养的方法 |
| CN110511907A (zh) * | 2019-05-16 | 2019-11-29 | 安徽瑞达健康产业有限公司 | 一种体外稳定扩增高纯度、高细胞毒活性nk细胞的方法 |
| CN111690610A (zh) * | 2020-07-22 | 2020-09-22 | 暨赛再生医学科技有限公司 | 一种高效诱导培养制备自然杀伤性nk细胞的方法 |
Non-Patent Citations (3)
| Title |
|---|
| STEFAN CARLENS ET AL.: "A New Method for In Vitro Expansion of Cytotoxic Human CD3-CD56+ Natural Killer Cells", 《HUMAN IMMUNOLOGY》, vol. 62, pages 1092, XP002276286, DOI: 10.1016/S0198-8859(01)00313-5 * |
| 申重阳等: "细胞因子预处理对体外扩增 NK 细胞活性的影响", 《中国现代医学杂志》, vol. 28, no. 28, pages 33 - 38 * |
| 邵鸿家等: "自然杀伤细胞分子生物学特征及体外扩增技术研究进展", 《医学综述》, vol. 18, no. 12, pages 1802 - 1805 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10806777B2 (en) | Method for allogeneic cell therapy | |
| CN102618498B (zh) | Hla-a0201限制性抗原特异性ctl制备方法 | |
| CN102575231B (zh) | 制备细胞因子诱导的杀伤细胞的方法 | |
| CN102597223A (zh) | 生产天然杀伤细胞的方法 | |
| US11566227B2 (en) | Kit containing medium for culturing natural killer cells and method of effectively culturing natural killer cells using the same | |
| CN115466726B (zh) | 一种nk细胞的高效基因转导方案 | |
| CN113046313A (zh) | 用于高效诱导扩增人体外周血杀伤性免疫细胞的组合物、试剂盒以及该免疫细胞的培养方法 | |
| CN113151168A (zh) | 一种人nk细胞培养体系及制备方法 | |
| CN102719402B (zh) | Hla-a0201限制性抗hpv抗原特异性ctl的制备方法 | |
| CN105524884A (zh) | Hla-a0201限制性抗afp抗原特异性ctl的制备方法 | |
| US10385315B2 (en) | Cells expressing Th1 characteristics and cytolytic properties | |
| CN102719400B (zh) | Hla-a0201限制性抗cea抗原特异性ctl的制备方法 | |
| CN109535241B (zh) | Dc-cik共培养细胞及其制备方法、致敏抗原和应用 | |
| CN110747167B (zh) | 一种半合子bak细胞的制备方法及其应用 | |
| CN116574679A (zh) | 特异性nk细胞的制备方法及其应用 | |
| CN113832101A (zh) | 自然杀伤细胞体外高效扩增的制备方法 | |
| CN117604011A (zh) | 融合基因、表达该融合基因的滋养型树突状细胞、自然杀伤细胞培养方法以及自然杀伤细胞 | |
| CN114657124A (zh) | 一种对肿瘤细胞具有高杀伤能力的复合型免疫细胞制备方法 | |
| Nhung | Efficient Expansion of Human Umbilical Cord Blood-Derived NK Cells Ex Vivo without Requiring Feeder Layers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20211224 |
|
| WW01 | Invention patent application withdrawn after publication |