[go: up one dir, main page]

CN108949696A - 免疫细胞培养基与其应用 - Google Patents

免疫细胞培养基与其应用 Download PDF

Info

Publication number
CN108949696A
CN108949696A CN201810955224.6A CN201810955224A CN108949696A CN 108949696 A CN108949696 A CN 108949696A CN 201810955224 A CN201810955224 A CN 201810955224A CN 108949696 A CN108949696 A CN 108949696A
Authority
CN
China
Prior art keywords
cell
car
culture
hydrochloride
immune cell
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
Application number
CN201810955224.6A
Other languages
English (en)
Inventor
吴建勇
易小萍
滕小锘
张大鹤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Misu Biotechnology Co Ltd
Original Assignee
Suzhou Misu Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Suzhou Misu Biotechnology Co Ltd filed Critical Suzhou Misu Biotechnology Co Ltd
Priority to CN201810955224.6A priority Critical patent/CN108949696A/zh
Publication of CN108949696A publication Critical patent/CN108949696A/zh
Pending legal-status Critical Current

Links

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
    • C12N5/0636—T lymphocytes
    • 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
    • C07K14/7051—T-cell receptor (TcR)-CD3 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/16—Magnesium; Mg chelators
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/22—Zinc; Zn chelators
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • 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
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2500/35—Polyols, e.g. glycerin, inositol
    • 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
    • C12N2500/36—Lipids
    • 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
    • C12N2500/38—Vitamins
    • 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
    • C12N2500/40—Nucleotides, nucleosides or bases
    • 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
    • C12N2500/46—Amines, e.g. putrescine
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • 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]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/71—Oxidoreductases (EC 1.)
    • 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
    • C12N2501/85—Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种免疫细胞培养基，其包含无水氯化钙等多种无机盐、L‑亮氨酸等多种氨基酸、白介素2、白介素4等组分。本发明的免疫细胞培养基在培养CAR‑T细胞时，能够显著提高细胞的增殖倍数，大幅延长细胞培养天数，并能够有效的保持表达表面标志物，同时其还具有保质期长，易于制备等优点。

Description

免疫细胞培养基与其应用
技术领域
本发明涉及一种免疫细胞培养基与其在培养CAR-T细胞中的应用，属于生物医疗技术领域。
背景技术
以嵌合抗原受体(chimericantigenreceptor，CAR)修饰的T细胞为代表的肿瘤靶向免疫治疗在体外和临床试验中表现出良好的靶向性、杀伤性和持久性，展示了巨大的应用潜力和发展前景。CAR-T细胞治疗的原理在于经嵌合抗原受体修饰的T细胞，可以特异性地识别肿瘤相关抗原，使效应T细胞的靶向性、杀伤活性和持久性均较常规应用的免疫细胞高，并可克服肿瘤局部免疫抑制微环境并打破宿主免疫耐受状态，即表达CAR的T细胞以抗原依赖、非MHC限制的方式结合肿瘤抗原，启动并活化特异性杀伤肿瘤反应。
CAR-T细胞治疗的一个重要环节是进行CAR-T细胞的扩增，而此环节中的一个非常关键的因素是选择有效的培养基。
迄今，有众多厂家发展了多种免疫细胞培养基，例如，如X-VIVO(Lonza出品)，KBM-551K、KBM-581、LONZA X-VIVO 15培养基(康宁出品)等。这些培养基产品目前已经被广泛的使用。但是，这些培养基都含有动物源成分，例如胎牛血清，这些动物源成分系从动物血清或组织中分离纯化出，可能含有动物病原体，因此有引起疾病的危险。
尽管如Gibco公司的AIM-V、Sigma公司的Stemline、Takara公司的GT-T551H3、Miltenyi公司的TexMACS等产品均声称是无血清培养基，但其在使用时，若非添加血清成分，则淋巴细胞的增殖较为缓慢，难以满足临床细胞治疗所需要的细胞数量。
此外，现有的免疫细胞培养基还存在保质期较短的缺陷，在培养CAR-T细胞的周期内，会在后期导致细胞增殖效率急剧下滑的问题。
发明内容
本发明的主要目的在于提供一种免疫细胞培养基与其应用，以克服现有技术中的不足。
为实现前述发明目的，本发明采用的技术方案包括：
本发明实施例提供了一种免疫细胞培养基，其包括：无水氯化钙112.5mg/L～115.8mg/L、L-亮氨酸57.85mg/L～58.95mg/L、五水硫酸铜0.0011mg/L～0.0012mg/L、L-赖氨酸盐酸盐92.20mg/L～93.25mg/L、硫辛酸0.101mg/L～0.108mg/L、九水硝酸铁0.04mg/L～0.05mg/L、L-蛋氨酸16.98mg/L～17.28mg/L、酚红7.8mg/L～8.3mg/L、七水硫酸亚铁0.410mg/L～0.418mg/L、L-苯丙氨酸35.42mg/L～36.55mg/L、1,4-丁二胺二盐酸盐0.078mg/L～0.080mg/L、氯化钾310.5mg/L～311.3mg/L、L-丝氨酸26.55mg/L～27.15mg/L、丙酮酸钠52mg/L～58mg/L、氯化镁28.61mg/L～28.38mg/L、L-苏氨酸53.41mg/L～53.49mg/L、维生素H 0.0025mg/L～0.0045mg/L、无水硫酸镁48.55mg/L～49.54mg/L、L-丙氨酸4.35mg/L～4.55mg/L、乙醇胺500.0mg/L～2000.0mg/L、D-泛酸钙2.21mg/L～2.26mg/L、氯化钠6950.5mg/L～7120.5mg/L、L-天门冬酰胺7.4mg/L～8.5mg/L、氯化胆碱8.88mg/L～9.18mg/L、转铁蛋白40～180mg/L、无水磷酸二氢钠54.15mg/L～54.55mg/L、.利托那韦200.0nmol/L～1000.0nmol/L、L-天门冬氨酸6.25mg/L～6.75mg/L、叶酸2.45mg/L～2.65mg/L、油酸100.0mg/L～1500.0mg/L、磷酸氢二钠70.52mg/L～73.22mg/L、L-半胱氨酸盐酸盐16.98mg/L～17.65mg/L、白介素410mg/L～80mg/L、i-肌醇12.2mg/L～12.7mg/L、人血清白蛋白500.0mg/L～3000.0mg/L、庆大霉素1×103～5×106U/L、七水硫酸锌56.431mg/L～210.433mg/L、重组人胰岛素0.5mg/L～5mg/L、L-谷氨酸7.25mg/L～7.55mg/L、抗坏血酸25.0mg/L～150.0mg/L、烟酰胺1.96mg/L～2.0mg/L、L-精氨酸盐酸盐145.5mg/L～148.5mg/L、L-脯氨酸17.15mg/L～17.25mg/L、IL-77μg/L～9μg/L、IL-153μg/L～5μg/L、亚油酸100.0mg/L～1200.0mg/L、盐酸吡哆醛1mg/L～2mg/L、孕酮8mg/L～60mg/L、L-胱氨酸盐酸盐30.19mg/L～32.29mg/L、L-色氨酸8.72mg/L～9.22mg/L、人过氧化氢酶200.0mg/L～2200.0mg/L、盐酸吡哆醇0.028mg/L～0.035mg/L、蛋氨酸脑啡肽385mg/L～425mg/L、干细胞生长因子5mg/L～40mg/L、L-酪氨酸38.2mg/L～39.4mg/L、核黄素0.213mg/L～0.219mg/L、甘氨酸18.35mg/L～19.25mg/L、L-缬氨酸52.45mg/L～53.65mg/L、白介素217～70mg/L、盐酸硫胺2.12mg/L～2.18mg/L、维生素C 0.5～5mg/L、L-组氨酸盐酸盐31.23mg/L～32.48mg/L、D-葡萄糖3110mg/L～3165mg/L、胸苷0.312mg/L～0.386mg/L、L-异亮氨酸54.21mg/L～54.83mg/L、次黄嘌呤1mg/L～3mg/L、维生素B120.61mg/L～0.72mg/L、NaHCO33.0mmol/L～29mmol/L。
本发明实施例还提供了所述的免疫细胞培养基在CAR-T细胞培养中的应用。
进一步地，所述CAR-T细胞选自CD19-CAR-T、CD20-CAR-T、K轻链-CAR-T、CD22-CAR-T、CD23-CAR-T、CD30-CAR-T或CD70-CAR-T。
本发明实施例还提供了一种CAR-T细胞培养方法，其包括：采用所述的免疫细胞培养基培养CAR-T细胞。
进一步地，所述的培养方法包括：将CAR-T细胞和CD3单克隆抗体加入所述的免疫细胞培养基中，调整细胞密度，进行细胞培养，培养72h后补加所述培养基和所述CD3单克隆抗体，之后继续培养4天以上，每隔2～3天补充所述免疫细胞培养基。
进一步地，所述细胞密度为(1～10)×106cell/mL。
进一步地，所述细胞培养的条件为5％CO2、37℃。
进一步地，所述CD3单克隆抗体的浓度为20～50ng/mL。
较之现有技术，本发明的免疫细胞培养基在培养CAR-T细胞时，能够显著提高细胞的增殖倍数，大幅延长细胞培养天数，并能够有效的保持表达表面标志物，同时其还具有保质期长，易于制备等优点。
附图说明
图1是分别采用本发明实施例1-实施例5、对照例1-对照例2的培养基进行免疫细胞培养的细胞增殖曲线图。
具体实施方式
鉴于现有技术存在的诸多缺陷，本案发明人经长期研究和大量实践，得以提出本发明的技术方案，其主要涉及一种改进的免疫细胞培养基及其在培养CAR-T细胞中的应用。
若非特殊说明，本说明书中所涉及的技术术语的释义与本领域的常规释义相同，例如：
嵌合抗原受体(CAR)：是CAR-T的核心部件，赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力，这使得经过CAR改造的T细胞相较于天然T细胞表面受体TCR能够识别更广泛的目标。CAR的基础设计中包括一个肿瘤相关抗原(tumor-associated antigen，TAA)结合区(通常来源于单克隆抗体抗原结合区域的scFV段)，一个胞外铰链区，一个跨膜区和一个胞内信号区。目标抗原的选择对于CAR的特异性、有效性以及基因改造T细胞自身的安全性来讲都是关键的决定因素。
CAR-T细胞：嵌合抗原受体T细胞。
CD19-CAR-T：靶向CD19分子的嵌合体抗原受体基因修饰的T细胞。
CD3：在免疫学中，CD3(分化簇3)T细胞的共受体是一种蛋白质复合物。在哺乳动物中，该复合物含有一个CD3γ链，CD3δ链，和2CD3ε链。这些链具有被称为一个分子副T细胞受体(TCR)和ζ-链以产生激活信号的T淋巴细胞。该TCR，ζ链和CD3分子一起构成的T细胞受体复合物。本发明的免疫细胞培养基中各原料均应是符合美国药典或《国家标准化学试剂》或《中华人民共和国药典》2010年版第二部的超纯或分析纯试剂，以满足临床安全合理的要求。
本发明的免疫细胞培养基不含任何动物源成份，其中的生物来源成分均为药物级或高度纯化的人源物质，因此安全性是有保证的。
本说明书中述及的免疫细胞培养基中各组分及细胞培养方法中所使用的细胞、原料均可由市场购得。
以下结合若干实施例对本发明的技术方案作更为具体的说明。
实施例1-实施例5的免疫细胞培养基的组分详见下表：
前述实施例1-实施例5中各免疫细胞培养基的制备方法包括：将上述培养基各组分与高纯灭菌水按比例混合，4℃震荡混匀1小时，然后通过0.22μm的滤膜过滤除菌待用。
采用实施例1-实施例5的免疫细胞培养基进行细胞培养，过程如下：将市购的表达嵌合抗原受体CD19CAR的T细胞用实施例1-实施例5的培养基重悬，同时加入30ng/mL的CD3单抗，调整接种密度1×106cell/mL，放入5％CO2，37℃培养箱中培养。培养72小时后按照5×105cell/mL的密度补加实施例1-实施例5的培养基，同时补加30ng/mL的CD3单抗。之后每2-3天补充该培养基一次，保持细胞密度在5×105cell/mL；之后，计算细胞的增殖曲线，并取培养到7天和21天的细胞进行流式检测。
细胞检测具体为：将收集到的细胞用PBS清洗2遍，通过流式仪检测其表面标志物CD4+，CD8+，以及CD19CAR的表达率。
另，分别采用X-VIVO、KBM-581培养基替代实施例1-实施例5的培养基，按照相同方式进行细胞培养，并采用相同方式进行测试，作为对比例1、2。
测试结果：
1、测得的细胞增殖曲线详见下图1。其中实施例1-5相应的曲线为命名为“实施例”的曲线(所用数据为平均测试数据)，对比例1、2相应曲线命名为“对比例1”、“对比例2”。
2、流式检测结果：
备注：上表中“实施例”相关数据为实施例1-实施例5所得数据的平均值。
显然，本发明实施例的免疫细胞培养基至少具有如下优点：能够大幅提高多种免疫细胞的增殖倍数，并可延长细胞培养天数，培养天数能够达到21天以上，且能非常好的保持表达CD4+、CD8+、CD19CAR等表面标志物。
应当理解，以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (8)

1.一种免疫细胞培养基，其特征在于包括：无水氯化钙112.5mg/L～115.8mg/L、L-亮氨酸57.85mg/L～58.95mg/L、五水硫酸铜0.0011mg/L～0.0012mg/L、L-赖氨酸盐酸盐92.20mg/L～93.25mg/L、硫辛酸0.101mg/L～0.108mg/L、九水硝酸铁0.04mg/L～0.05mg/L、L-蛋氨酸16.98mg/L～17.28mg/L、酚红7.8mg/L～8.3mg/L、七水硫酸亚铁0.410mg/L～0.418mg/L、L-苯丙氨酸35.42mg/L～36.55mg/L、1,4-丁二胺二盐酸盐0.078mg/L～0.080mg/L、氯化钾310.5mg/L～311.3mg/L、L-丝氨酸26.55mg/L～27.15mg/L、丙酮酸钠52mg/L～58mg/L、氯化镁28.61mg/L～28.38mg/L、L-苏氨酸53.41mg/L～53.49mg/L、维生素H 0.0025mg/L～0.0045mg/L、无水硫酸镁48.55mg/L～49.54mg/L、L-丙氨酸4.35mg/L～4.55mg/L、乙醇胺500.0mg/L～2000.0mg/L、D-泛酸钙2.21mg/L～2.26mg/L、氯化钠6950.5mg/L～7120.5mg/L、L-天门冬酰胺7.4mg/L～8.5mg/L、氯化胆碱8.88mg/L～9.18mg/L、转铁蛋白40～180mg/L、无水磷酸二氢钠54.15mg/L～54.55mg/L、.利托那韦200.0nmol/L～1000.0nmol/L、L-天门冬氨酸6.25mg/L～6.75mg/L、叶酸2.45mg/L～2.65mg/L、油酸100.0mg/L～1500.0mg/L、磷酸氢二钠70.52mg/L～73.22mg/L、L-半胱氨酸盐酸盐16.98mg/L～17.65mg/L、白介素410mg/L～80mg/L、i-肌醇12.2mg/L～12.7mg/L、人血清白蛋白500.0mg/L～3000.0mg/L、庆大霉素1×103～5×106U/L、七水硫酸锌56.431mg/L～210.433mg/L、重组人胰岛素0.5mg/L～5mg/L、L-谷氨酸7.25mg/L～7.55mg/L、抗坏血酸25.0mg/L～150.0mg/L、烟酰胺1.96mg/L～2.0mg/L、L-精氨酸盐酸盐145.5mg/L～148.5mg/L、L-脯氨酸17.15mg/L～17.25mg/L、IL-77μg/L～9μg/L、IL-153μg/L～5μg/L、亚油酸100.0mg/L～1200.0mg/L、盐酸吡哆醛1mg/L～2mg/L、孕酮8mg/L～60mg/L、L-胱氨酸盐酸盐30.19mg/L～32.29mg/L、L-色氨酸8.72mg/L～9.22mg/L、人过氧化氢酶200.0mg/L～2200.0mg/L、盐酸吡哆醇0.028mg/L～0.035mg/L、蛋氨酸脑啡肽385mg/L～425mg/L、干细胞生长因子5mg/L～40mg/L、L-酪氨酸38.2mg/L～39.4mg/L、核黄素0.213mg/L～0.219mg/L、甘氨酸18.35mg/L～19.25mg/L、L-缬氨酸52.45mg/L～53.65mg/L、白介素217～70mg/L、盐酸硫胺2.12mg/L～2.18mg/L、维生素C 0.5～5mg/L、L-组氨酸盐酸盐31.23mg/L～32.48mg/L、D-葡萄糖3110mg/L～3165mg/L、胸苷0.312mg/L～0.386mg/L、L-异亮氨酸54.21mg/L～54.83mg/L、次黄嘌呤1mg/L～3mg/L、维生素B120.61mg/L～0.72mg/L、NaHCO33.0mmol/L～29mmol/L。
2.权利要求1所述的免疫细胞培养基在CAR-T细胞培养中的应用。
3.如权利要求2所述的应用，其特征在于：所述CAR-T细胞选自CD19-CAR-T、CD20-CAR-T、K轻链-CAR-T、CD22-CAR-T、CD23-CAR-T、CD30-CAR-T或CD70-CAR-T。
4.一种CAR-T细胞培养方法，其特征在于包括：采用如权利要求1所述的免疫细胞培养基培养CAR-T细胞。
5.根据权利要求6所述的培养方法，其特征在于包括：将CAR-T细胞和CD3单克隆抗体加入所述的免疫细胞培养基中，调整细胞密度，进行细胞培养，培养72h后补加所述培养基和所述CD3单克隆抗体，之后继续培养4天以上，每隔2～3天补充所述免疫细胞培养基。
6.根据权利要求5所述的培养方法，其特征在于，所述细胞密度为(1～10)×106cell/mL。
7.根据权利要求5所述的培养方法，其特征在于，所述细胞培养的条件为5％CO2、37℃。
8.根据权利要求5所述的培养方法，其特征在于，所述CD3单克隆抗体的浓度为20～50ng/mL。
CN201810955224.6A 2018-08-21 2018-08-21 免疫细胞培养基与其应用 Pending CN108949696A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810955224.6A CN108949696A (zh) 2018-08-21 2018-08-21 免疫细胞培养基与其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810955224.6A CN108949696A (zh) 2018-08-21 2018-08-21 免疫细胞培养基与其应用

Publications (1)

Publication Number Publication Date
CN108949696A true CN108949696A (zh) 2018-12-07

Family

ID=64473335

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810955224.6A Pending CN108949696A (zh) 2018-08-21 2018-08-21 免疫细胞培养基与其应用

Country Status (1)

Country Link
CN (1) CN108949696A (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110184239A (zh) * 2019-06-12 2019-08-30 兰莲(杭州)生物科技有限公司 一种活性化t淋巴细胞培养液及其活性化t淋巴细胞的制备方法
CN110894466A (zh) * 2019-12-20 2020-03-20 广州海润康华生物科技有限公司 一种干细胞培养用培养基
CN117070454A (zh) * 2023-10-18 2023-11-17 北京细胞治疗集团有限公司 一种car-t细胞的制备方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014165707A2 (en) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Effective generation of tumor-targeted t-cells derived from pluripotent stem cells
CN104450614A (zh) * 2014-12-05 2015-03-25 上海安集协康生物技术有限公司 一种无动物蛋白源的免疫细胞无血清培养基及其使用方法
CN104818248A (zh) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 一种免疫细胞培养基、培养方法及应用
CN105154402A (zh) * 2015-10-28 2015-12-16 广州赛莱拉干细胞科技股份有限公司 一种培养基及其应用、培养方法
CN105176925A (zh) * 2015-09-26 2015-12-23 友康恒业生物科技（北京）有限公司 一种免疫细胞无血清培养基及其制备和用途
CN106834229A (zh) * 2017-01-25 2017-06-13 华东理工大学 用于人免疫杀伤细胞体外扩增的无血清培养基
WO2017219528A1 (zh) * 2016-06-21 2017-12-28 林志国 特异性识别EGFRvⅢ的含CD70嵌合抗原受体修饰T细胞的制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014165707A2 (en) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Effective generation of tumor-targeted t-cells derived from pluripotent stem cells
CN104450614A (zh) * 2014-12-05 2015-03-25 上海安集协康生物技术有限公司 一种无动物蛋白源的免疫细胞无血清培养基及其使用方法
CN104818248A (zh) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 一种免疫细胞培养基、培养方法及应用
CN105176925A (zh) * 2015-09-26 2015-12-23 友康恒业生物科技（北京）有限公司 一种免疫细胞无血清培养基及其制备和用途
CN105154402A (zh) * 2015-10-28 2015-12-16 广州赛莱拉干细胞科技股份有限公司 一种培养基及其应用、培养方法
WO2017219528A1 (zh) * 2016-06-21 2017-12-28 林志国 特异性识别EGFRvⅢ的含CD70嵌合抗原受体修饰T细胞的制备方法
CN106834229A (zh) * 2017-01-25 2017-06-13 华东理工大学 用于人免疫杀伤细胞体外扩增的无血清培养基

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALNABHAN, R等: "Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells", 《CYTOTHERAPY》 *
CHAVEZ, AT等: "Impact of Different Culture Media Supplements on CAR T Cell Function", 《MOLECULAR THERAPY》 *
PTACKOVA, P等: "A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21", 《CYTOTHERAPY》 *
高群等: "不同培养基与血清的培养体系对细胞因子诱导杀伤细胞增殖和功能的影响", 《中国肿瘤生物治疗杂志》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110184239A (zh) * 2019-06-12 2019-08-30 兰莲(杭州)生物科技有限公司 一种活性化t淋巴细胞培养液及其活性化t淋巴细胞的制备方法
CN110184239B (zh) * 2019-06-12 2021-04-06 蓝莲(杭州)生物科技有限公司 一种活性化t淋巴细胞培养液及其活性化t淋巴细胞的制备方法
CN110894466A (zh) * 2019-12-20 2020-03-20 广州海润康华生物科技有限公司 一种干细胞培养用培养基
CN117070454A (zh) * 2023-10-18 2023-11-17 北京细胞治疗集团有限公司 一种car-t细胞的制备方法

Similar Documents

Publication Publication Date Title
Stremenova Spegarova et al. Germline TET2 loss of function causes childhood immunodeficiency and lymphoma
Nakabayashi et al. Growth of human hepatoma cell lines with differentiated functions in chemically defined medium
Zuckerman et al. Long-term human peripheral blood monocyte cultures: establishment, metabolism and morphology of primary human monocyte-macrophage cell cultures
CN105462924B (zh) Nk细胞的培养方法及无血清培养基组合
Miller et al. Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.
CN104450614B (zh) 一种无动物蛋白源的免疫细胞无血清培养基及其使用方法
JP3410103B2 (ja) シュワン細胞の分離および培養
CN108949696A (zh) 免疫细胞培养基与其应用
CN1272136A (zh) 体外造血细胞的刺激
EP0136353A1 (fr) Milieu de culture de cellules animales sans serum et procedes de culture primaire et d'obtention de lignees cellulaires utilisant ce milieu.
JPS63267269A (ja) 細胞培養のための基礎栄養培地
CN106834229B (zh) 用于人免疫杀伤细胞体外扩增的无血清培养基
US6033660A (en) Method of treating a nervous system injury with cultured schwann cells
BRPI0707529A2 (pt) métodos para modulação do teor de manose de proteìnas recombinantes
CN102676453A (zh) 一种nk和/或nkt细胞的培养方法
JPS62126995A (ja) ヒト抗体の製造方法
Franěk et al. Specific effects of synthetic oligopeptides on cultured animal cells
CN107164322A (zh) 一种用于人γδT细胞扩增的培养基及其制备方法
CN103834615A (zh) 一种适用于培养γδT细胞的无血清培养基
CN110760481B (zh) 一种改良rpmi1640培养基及其应用
JP2022510387A (ja) ガンマデルタｔ細胞集団の選択的生体内増殖方法及びその組成物
Goss Characterization of cystathionine synthase as a selectable, liver-specific trait in rat hepatomas
Caviedes et al. Studies on a cell line from a functional rat thyroid tumor in continuous culture
JP4510425B2 (ja) 三次元培養による細胞の培養方法
Schimmer et al. Glial tumor cells deficient in thymidine kinase: isolation and characterization

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
RJ01 Rejection of invention patent application after publication

Application publication date: 20181207

RJ01 Rejection of invention patent application after publication