CN113755480B - 病毒固相感染体外培养细胞的方法 - Google Patents
病毒固相感染体外培养细胞的方法 Download PDFInfo
- Publication number
- CN113755480B CN113755480B CN202110299370.XA CN202110299370A CN113755480B CN 113755480 B CN113755480 B CN 113755480B CN 202110299370 A CN202110299370 A CN 202110299370A CN 113755480 B CN113755480 B CN 113755480B
- Authority
- CN
- China
- Prior art keywords
- virus
- cell
- cells
- solid phase
- dmem
- 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.)
- Active
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 102
- 239000007790 solid phase Substances 0.000 title claims abstract description 43
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000000338 in vitro Methods 0.000 title claims abstract description 8
- 239000000725 suspension Substances 0.000 claims abstract description 12
- 230000001464 adherent effect Effects 0.000 claims abstract description 10
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 6
- 108010010803 Gelatin Proteins 0.000 claims abstract description 6
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims abstract description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 6
- 239000008273 gelatin Substances 0.000 claims abstract description 6
- 229920000159 gelatin Polymers 0.000 claims abstract description 6
- 235000019322 gelatine Nutrition 0.000 claims abstract description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 6
- 229920000656 polylysine Polymers 0.000 claims abstract description 6
- 238000011081 inoculation Methods 0.000 claims abstract description 5
- 229940069078 citric acid / sodium citrate Drugs 0.000 claims abstract description 3
- 238000004113 cell culture Methods 0.000 claims description 20
- 238000012258 culturing Methods 0.000 claims description 14
- 239000011521 glass Substances 0.000 claims description 13
- 238000011534 incubation Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 239000006285 cell suspension Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 238000010899 nucleation Methods 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 239000005723 virus inoculator Substances 0.000 claims description 6
- 230000001678 irradiating effect Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000010561 standard procedure Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 79
- 238000011160 research Methods 0.000 abstract description 13
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 230000009385 viral infection Effects 0.000 abstract description 8
- 238000002659 cell therapy Methods 0.000 abstract description 4
- 210000004748 cultured cell Anatomy 0.000 abstract description 3
- 210000000130 stem cell Anatomy 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 description 10
- 230000001404 mediated effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000003013 cytotoxicity Effects 0.000 description 5
- 231100000135 cytotoxicity Toxicity 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 241000701161 unidentified adenovirus Species 0.000 description 5
- 239000000834 fixative Substances 0.000 description 4
- 238000009169 immunotherapy Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000969 carrier Substances 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003151 transfection method Methods 0.000 description 2
- 238000011523 CAR-T cell immunotherapy Methods 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000013603 viral vector Substances 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/096—Polyesters; Polyamides
-
- 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
-
- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle 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
- 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
- C12N5/0693—Tumour cells; Cancer 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
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Reproductive Health (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及到分子生物学、细胞生物学等生命科学基础研究及细胞治疗应用领域,具体的说是一种在低病毒滴度条件下实现理想的细胞感染效率的病毒固相感染体外培养细胞的方法,其特征在于,包括配置病毒固定液、固定病毒和细胞接种三个步骤,其中所述病毒固定液包括以下各组分:柠檬酸/柠檬酸钠、明胶、海藻糖、左旋多聚赖氨酸、PEG‑20000、DMEM、FBS;本发明可以在不增加病毒用量的前提下提高悬浮细胞的感染率,在细胞治疗领域具有重要的应用价值;而且可以实现“芯片”模式的高通量研究,即在同一培养单位内可以研究多种基因(适用于贴壁细胞),在提高病毒感染效率的同时,也极大降低了病毒和细胞的用量,特别是针对珍贵的原代细胞及干细胞。
Description
技术领域:
本发明涉及到分子生物学、细胞生物学等生命科学基础研究及细胞治疗应用领域,具体的说是一种在低病毒滴度条件下实现理想的细胞感染效率的病毒固相感染体外培养细胞的方法。
背景技术:
基因转染是一种“将具生物功能的核酸转移或运送到细胞内并使核酸在细胞内维持其生物功能”的技术,已广泛应用于基因组功能研究(基因表达调控,基因功能,信号转导和药物筛选研究)和基因治疗研究(如CAR-T细胞免疫治疗)。
随着基因与蛋白功能研究的深入,转染已成为实验室工作中经常涉及的基本方法。转染大致可分为物理介导、化学介导和生物介导三类途径。电穿孔法、显微注射和基因枪属于通过物理方法将基因导入细胞的范例;化学介导方法很多,如经典的磷酸钙共沉淀法、脂质体转染方法、和多种阳离子物质介导的技术;生物介导方法,有较为原始的原生质体转染和现在比较多见的各种病毒介导的转染技术。理想细胞转染方法,应该具有转染效率高、细胞毒性小等优点。病毒介导的转染技术,是目前转染效率最高的方法,同时具有细胞毒性低的优势。但是,病毒转染方法对细胞类型有很强的选择性,比如病毒对悬浮细胞感染效率偏低,要达到理想的感染率,需要提高病毒滴度,但是高病毒滴度会增加细胞毒性以及成本。
细胞免疫治疗是近几年兴起的疾病治疗新技术,是指利用某些具有特定功能的细胞的特性,采用生物工程方法获取和/或通过体外扩增、特殊培养等处理后,产生的特异性功能强大的细胞,回输体内从而达到治疗疾病的目的。其中最典型目前相对成熟的就是CAR-T疗法,CAR-T疗法就是嵌合抗原受体T细胞免疫疗法,是一种治疗肿瘤的新型精准靶向疗法,近几年通过优化改良在临床肿瘤治疗上取得很好的效果,是一种非常有前景的,能够精准、快速、高效,且有可能治愈癌症的新型肿瘤免疫治疗方法。腺病毒(adenovirus,Adv)为细胞免疫治疗中报告最多的一种病毒载体,其体外制备滴度较大,装载外源基因容量最大达35kb,不整合入宿主细胞基因组因而避免插入突变的危险,能感染分裂细胞和非分裂细胞。
通过以上描述可看出,病毒感染培养细胞技术在生命医学领域广泛应用,所以提高病毒的感染效率至关重要。以生物医学研究常用的慢病毒、腺病毒为例,其对贴壁生长的传代细胞的感染效率较高,常规的感染操作即可满足相关的研究应用。但对常规的病毒感染操作对悬浮细胞感染效率较低,极大的限制了悬浮细胞的研究和临床应用,特别是在CAR-T领域,很大程度限制了临床治疗的成功率,故探索新的提高病毒感染效率的方法意义尤为重大。
常规的病毒感染培养细胞操作是,先将细胞接种至培养载体上,再将病毒加入细胞中使病毒进入细胞的过程。该过程病毒对对悬浮细胞感染效率较低,如要提高细胞的病毒效率,必须提高病毒用量,而高病毒滴度不但增加了科研或治疗成本,也对细胞产生一定的细胞毒性。所以在低病毒滴度条件下实现理想的细胞感染效率,是本发明要解决的问题。
发明内容:
与现有技术中的病毒感染方式不同,本发明通过特殊的病毒固定液及固定工艺,将病毒固定在固相载体上,本发明不但能保证病毒固定后的活性,而且极大提高了病毒的感染效率,实现低病毒滴度条件下的高细胞感染率。该技术不但提高了悬浮细胞的感染效率,而且可以对贴壁细胞进行高通量的研究。
本发明通过以下措施达到:
一种病毒固相感染体外培养细胞的方法,其特征在于,包括配置病毒固定液、固定病毒和细胞接种三个步骤,其中所述病毒固定液包括以下各组分:
柠檬酸/柠檬酸钠、明胶、海藻糖、左旋多聚赖氨酸、PEG-20000、DMEM、FBS;
所述固定病毒包括以下步骤:
步骤1:固相载体预处理,将固相载体平放置于生物安全柜中,打开安全柜紫外灯,紫外照射30分钟,所用固相载体为细胞培养微孔板或载玻片;
步骤2:病毒配制,用病毒固定缓冲液,稀释预固定的病毒,所用病毒浓度为103vp/μL-105vp/μL,并充分的混匀;
步骤3:固定病毒,将步骤2中配好的病毒溶液固定在固相载体上,固定完毕后,自然晾干,密封,并4℃储存备用。
本发明步骤3中,若对于贴壁细胞,执行以下步骤:
用2μL量程的微量移液枪配低吸附的无菌枪头,调整取样量为0.2μL,取0.2μL步骤2中的病毒液,垂直点在固相载体上,形成病毒斑,此病毒斑直径范围为1.0-2.0mm,优选为1.5mm,病毒斑之间的间距控制为2-3mm,病毒接种完毕后,置于生物安全柜中,自然晾干30分钟,然后密封,并4℃储存备用。
本发明步骤3中,若对于悬浮细胞,执行以下步骤:用无菌接种棒将病毒液均匀的涂在固相载体表面,接种用量为12μL/cm2,病毒接种完毕后,于生物安全柜中,自然晾干30分钟,然后密封,并4℃储存备用。
本发明所述细胞接种通过以下步骤实现:
对于贴壁细胞-6孔板固相载体,执行以下步骤:
步骤4-1:用DMEM-10%FBS培养基调整细胞浓度为1*104/mL;
步骤4-2:每孔加入2mL细胞悬液,即细胞接种总量为2*104,于细胞培养箱中培养;
步骤4-3:培养12小时后,标准操作更换细胞培养液,继续培养。
对于贴壁细胞-载玻片固相载体,执行以下步骤:
步骤5-1:将固定病毒的载玻片,在无菌条件下,病毒面向上,放置在直径10CM宽的细胞培养皿中;
步骤5-2:用DMEM-10%FBS培养基调整细胞浓度为1*104/mL,加入10mL DMEM-10%FBS细胞悬液,即细胞接种总量为1*105,于细胞培养箱中静置培养;
步骤5-3:培养12小时后,无菌条件下取出载玻片,转入另一装有10mL DMEM-10%FBS细胞培养液的培养皿中,继续培养。
对于悬浮细胞-6孔板固相载体,执行以下步骤:
步骤6-1:用DMEM-10%FBS培养基调整细胞浓度为5*104/mL;
步骤6-2:每孔加入2mL细胞悬液,即细胞接种总量为1*105,于细胞培养箱中培养;
步骤6-3:培养6小时后,标准操作更换细胞培养液,转移至10CM宽的细胞培养皿中继续培养,培养液采用10mL/DMEM-10%FBS。
本发明可以在不增加病毒用量的前提下提高悬浮细胞的感染率,在细胞治疗领域具有重要的应用价值;而且可以实现“芯片”模式的高通量研究,即在同一培养单位内可以研究多种基因(适用于贴壁细胞),在提高病毒感染效率的同时,也极大降低了病毒和细胞的用量,特别是针对珍贵的原代细胞及干细胞。
附图说明:
附图1是本发明中不同固相载体(载玻片和微孔板)上腺病毒-GFP和慢病毒-GFP固相感染Hela细胞。
附图2是本发明中不同滴度的腺病毒-GFP固相感染Hela细胞(微孔板)。
附图3是本发明中腺病毒-GFP固定后储存稳定性验证,感染Hela细胞(微孔板)。
附图4是本发明中低病毒滴度固相感染和常规感染对不同细胞的感染率对比(腺病毒-GFP)。
具体实施方式:
下面结合附图和实施例对本发明作进一步的说明:
实施案例1:
如附图1、附图2和附图3所示,本发明提出了一种适合贴壁细胞固相感染的制备方法,其特征在于包括以下步骤:
步骤1:按照如下比例配置病毒固定液,取0.1M-pH6.6柠檬酸钠、0.1%明胶、4.5%海藻糖、0.1%左旋多聚赖氨酸、0.5%PEG-20000、10%FBS,上述各组分均用DMEM培养基配制,并用0.2μm过滤器,固定液过滤除菌,4℃储存备用;
步骤2:固相载体预处理,将细胞培养板或载玻片等固相载体平放置于生物安全柜中,打开安全柜紫外灯,紫外照射30分钟;
步骤3:病毒配制:用病毒固定液稀释预固定的腺病毒-GFP或慢病毒-GFP,所用病毒终浓度为105vp/μL,并充分的混匀;
步骤4:病毒固定:用2μL量程的微量移液枪配低吸附的无菌枪头,调整取样量为0.2μL,取0.2μL步骤3中的病毒液,垂直点在固相载体上,形成病毒斑,此病毒斑直径大约为1.5mm,病毒斑之间的间距控制为2-3mm,病毒接种完毕后,静置于生物安全柜中,自然晾干30分钟,然后密封,并4℃储存备用;
步骤5-1:接种细胞(6孔板),用DMEM-10%FBS培养基调整Hela细胞浓度为1*104/mL;每孔加入2mL细胞悬液,即细胞接种总量为2*104,于细胞培养箱中培养。培养12小时后,标准操作更换细胞培养液,继续培养;
步骤5-2:接种细胞(载玻片),将固定病毒的载玻片,在无菌条件下,病毒面向上,放置在直径10CM宽的细胞培养皿中,用DMEM-10%FBS培养基调整细胞浓度为1*104/mL,加入10mL DMEM-10%FBS细胞悬液,于细胞培养箱中静置培养,培养12小时后,无菌条件下取出载玻片,转入另一装有10mL DMEM-10%FBS细胞培养液的培养皿中,继续培养。
结果分析:
附图1所示,本发明分别选用转有GFP的腺病毒和慢病毒两种常用的病毒载体,分别用细胞培养板和载玻片为固相载体,固相感染Hela细胞48小时后,于荧光显微镜下观察感染细胞的情况。结果显示,该技术感染效果极佳,而且在不同的固相载体上呈现不同的形状,细胞培养板呈现空心环状、载玻片呈现实心的点状。而且细胞斑之间间隙清晰,无明显扩散,故该技术可以用于单培养单位内多病毒或多基因的高通量研究。
附图2所示,按照上述工艺固定不同病毒滴度的腺病毒-GFP,感染Hela细胞,48h后荧光显微镜下观察,结果表明在低病毒滴度103vp/μL的条件下,仍能有效感染细胞。随着病毒滴度的提高,GFP表达明显增加,提示该技术良好的固相感染性能。
附图3所示,病毒在固相载体固定并冷藏干燥条件下储存,分别在储存1周至6个月的时间内取出,感染Hela细胞验证病毒的感染活性,结果显示,该方法制备的病毒固相感染产品具有良好的储存稳定性。
本发明提出了一种新型的病毒固相感染技术,通过特制的病毒缓冲液,将病毒固定在固相载体上。该固相载体可以为细胞培养常用的培养板,也可以实验室常用的玻璃载玻片,具体可根据研究的需要选用。该技术通过特殊的工艺处理,可以确保固定病毒在干燥条件下良好的病毒活性。而且病毒无明显扩散,细胞斑之间间隙清晰,故该技术可以用于单培养单位内多病毒或多基因的高通量研究。
实施案例2:如附图4所示,本发明提出了一种低病毒滴度条件下提高细胞感染效率的固相感染的制备方法,其特征在于包括以下步骤:
步骤1:按照如下比例配置病毒固定液,0.1M-pH6.6柠檬酸钠、0.1%明胶、4.5%海藻糖、0.1%左旋多聚赖氨酸、0.5%PEG-20000、10%FBS,上述各组分均用标准配方的DMEM培养基配制,并用0.2μm过滤器,固定液过滤除菌,4℃储存备用。
步骤2:固相载体预处理,将6孔板做为固相载体,平放置于生物安全柜中,打开安全柜紫外灯,紫外照射30分钟;
步骤3:病毒配制,用病毒固定液稀释预固定的腺病毒-GFP所用病毒终浓度为103vp/μL,并充分的混匀;
步骤4:病毒固定,用无菌接种棒将病毒液均匀的涂在固相载体表面,接种用量为12μL/cm2。病毒接种完毕后,于生物安全柜中,自然晾干30分钟。然后密封,并4℃储存备用
步骤5:接种细胞。用DMEM-10%FBS培养基调整Hela、A549、7402和K562细胞浓度为均为1*104/mL;每孔分别加入2mL细胞悬液,即细胞接种总量为2*104,于细胞培养箱中培养。培养12小时后,标准操作更换细胞培养液,继续培养。同时,对正常培养的四种细胞,按照同规格细胞接种量正常接种培养并加入病毒,正常感染并培养。培养48小时后,荧光显微镜下观察感染效率。
结果分析:
附图4显示,在低病毒滴度条件下,四种不同的细胞,相比常规操作方式,该固相感染技术均具有极高的感染效率,特别是对K562悬浮细胞的感染效率有非常明显的提高。
本发明提出的固相感染细胞的方法,可以在低病毒滴度条件下对细胞具有较高的感染效率。低病毒滴度,不仅可以降低细胞毒性,而且可以极大的降低病毒成本。
Claims (1)
1.一种病毒固相感染体外培养细胞的方法,其特征在于,包括配置病毒固定液、固定病毒和细胞接种三个步骤,其中所述病毒固定液包括以下各组分:
柠檬酸/柠檬酸钠、明胶、海藻糖、左旋多聚赖氨酸、PEG-20000、DMEM、FBS,具体为:取0.1M-pH6 .6柠檬酸钠、0.1%明胶、4.5%海藻糖、0.1%左旋多聚赖氨酸、0.5%PEG-20000、10%FBS,上述各组分均用DMEM培养基配制,并用0.2μm过滤器,固定液过滤除菌,4℃储存备用;
所述固定病毒包括以下步骤:
步骤1:固相载体预处理,将固相载体平放置于生物安全柜中,打开安全柜紫外灯,紫外照射30分钟,所用固相载体为细胞培养微孔板或载玻片;
步骤2:病毒配制, 用病毒固定缓冲液,稀释预固定的病毒,所用病毒浓度为103vp/μL-105vp/μL,并充分的混匀;
步骤3:固定病毒,将步骤2中配好的病毒溶液固定在固相载体上,固定完毕后,自然晾干,密封,并4℃储存备用;
步骤3中,若对于贴壁细胞,执行以下步骤:
用2μL量程的微量移液枪配低吸附的无菌枪头,调整取样量为0.2μL,取0.2μL步骤2中的病毒液,垂直点在固相载体上,形成病毒斑,此病毒斑直径范围为1.0-2.0mm,病毒斑之间的间距控制为2-3mm,病毒接种完毕后,于生物安全柜中,自然晾干30分钟,然后密封,并4℃储存备用;
步骤3中,若对于悬浮细胞,执行以下步骤:用无菌接种棒将病毒液均匀的涂在固相载体表面,接种用量为12μL/cm2,病毒接种完毕后,于生物安全柜中,自然晾干30分钟,然后密封,并4℃储存备用;
所述细胞接种通过以下步骤实现:
对于贴壁细胞-6孔板固相载体,执行以下步骤:
步骤4-1:用DMEM-10%FBS培养基调整细胞浓度为1*104/mL;
步骤4-2:每孔加入2mL细胞悬液,即细胞接种总量为2*104,于细胞培养箱中培养;
步骤4-3:培养12小时后,标准操作更换细胞培养液,继续培养;
对于贴壁细胞-载玻片固相载体,执行以下步骤:
步骤5-1:将固定病毒的载玻片,在无菌条件下,病毒面向上,放置在直径10CM宽的细胞培养皿中;
步骤5-2:用DMEM-10%FBS培养基调整细胞浓度为1*104/mL,加入10mL DMEM-10%FBS细胞悬液,即细胞接种总量为1*105,于细胞培养箱中静置培养;
步骤5-3:培养12小时后,无菌条件下取出载玻片,转入另一装有10mL DMEM-10%FBS细胞培养液的培养皿中,继续培养;
对于悬浮细胞-6孔板固相载体,执行以下步骤:
步骤6-1:用DMEM-10%FBS培养基调整细胞浓度为5*104/mL;
步骤6-2:每孔加入2mL细胞悬液,即细胞接种总量为1*105,于细胞培养箱中培养;
步骤6-3:培养6小时后,标准操作更换细胞培养液,转移至10CM宽的细胞培养皿中继续培养,培养液采用10mL/DMEM-10%FBS。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110299370.XA CN113755480B (zh) | 2021-03-19 | 2021-03-19 | 病毒固相感染体外培养细胞的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110299370.XA CN113755480B (zh) | 2021-03-19 | 2021-03-19 | 病毒固相感染体外培养细胞的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113755480A CN113755480A (zh) | 2021-12-07 |
CN113755480B true CN113755480B (zh) | 2023-12-05 |
Family
ID=78786760
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110299370.XA Active CN113755480B (zh) | 2021-03-19 | 2021-03-19 | 病毒固相感染体外培养细胞的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113755480B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115386595A (zh) * | 2022-09-05 | 2022-11-25 | 安徽大学 | 一种感染细胞的方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0209680D0 (en) * | 2002-04-27 | 2002-06-05 | Univ Strathclyde | Immobilisation and stabilisation of bacteriophage |
WO2004078906A2 (de) * | 2003-03-04 | 2004-09-16 | NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen | Verfahren zur herstellung eines trägers mit immobilisierten viren und verwendung eines derartigen trägers |
EP3403671B1 (en) * | 2016-01-15 | 2023-06-07 | KM Biologics Co., Ltd. | Vaccine containing immobilized virus particles |
-
2021
- 2021-03-19 CN CN202110299370.XA patent/CN113755480B/zh active Active
Non-Patent Citations (1)
Title |
---|
Immobilization and One-Dimensional Arrangement of Virus Capsids with Nanoscale Precision Using DNA Origami;Nicholas Stephanopoulos等;Nano Lett.;第10卷;第2714-2720页 * |
Also Published As
Publication number | Publication date |
---|---|
CN113755480A (zh) | 2021-12-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR102022952B1 (ko) | 백시니아 바이러스의 생산을 위한 방법 및 조성물 | |
RU2706693C2 (ru) | Вирусная вакцина и способы ее производства | |
CN113755480B (zh) | 病毒固相感染体外培养细胞的方法 | |
CN107190024A (zh) | 一种稳定表达ns1蛋白细胞株的构建方法 | |
CN1657632A (zh) | 表达人类cyp2j2反义基因的重组腺相关病毒及其制备方法 | |
CN114561354B (zh) | 一种提高nk细胞杀伤活性的方法 | |
CN103160478A (zh) | 用生物反应器扩增重组腺病毒的优化工艺方法 | |
Cervera et al. | Large-scale transient transfection of suspension mammalian cells for VLP production | |
CN109868260A (zh) | 一种car-t细胞的制备方法 | |
CN112094814B (zh) | 一种通过灌流培养工艺制备腺病毒载体疫苗的方法 | |
US9932562B2 (en) | Drain down and re-feed of microcarrier bioreactor | |
CN1134537C (zh) | 人原始间叶干细胞群的制备方法 | |
CN116656628A (zh) | 一种痘病毒载体疫苗的制备方法 | |
CN111420031A (zh) | 一种经基因工程改造的抗肿瘤微颗粒的制备方法及应用 | |
CN116478925A (zh) | 一种体外快速制备肿瘤细胞球的方法 | |
CN105233303B (zh) | 一种通过基因改造血管内皮细胞促进创面愈合的方法及其应用 | |
CN109331171A (zh) | 一种疟原虫蛋白的制备方法及其在抗肿瘤方面的应用 | |
CN109453366A (zh) | 一种抗肿瘤蛋白质的制备方法及用途 | |
JPH11243948A (ja) | 動物細胞増殖用の細胞培養床基材及びその調製方法 | |
CN107432931A (zh) | 一种微载体培养水痘病毒制备疫苗的方法 | |
CN115141273A (zh) | 一种猫杯状病毒的单克隆抗体及其应用 | |
CN111378690B (zh) | 一种嵌合抗原受体t细胞的制备方法 | |
US20200040063A1 (en) | Process of cloning and further purification to make a recombinant intravenous immunoglobulin | |
CN116694580A (zh) | 一种生产重组腺病毒的方法 | |
CN112695032B (zh) | 一种启动子pLRRK2及其应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |