CN108865989A - 一种脐带间充质干细胞的培养基 - Google Patents

一种脐带间充质干细胞的培养基 Download PDF

Info

Publication number
CN108865989A
CN108865989A CN201810813395.5A CN201810813395A CN108865989A CN 108865989 A CN108865989 A CN 108865989A CN 201810813395 A CN201810813395 A CN 201810813395A CN 108865989 A CN108865989 A CN 108865989A
Authority
CN
China
Prior art keywords
culture medium
growth factor
umbilical cord
stem cells
mesenchymal stem
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
CN201810813395.5A
Other languages
English (en)
Inventor
宫丽
董振磊
张炳强
戚凤春
章宏
金铎
于淼
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.)
QINGDAO RE-STORE BIOTECHNOLOGY Co.,Ltd.
Original Assignee
Jilin Jihui 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 Jilin Jihui Biotechnology Co Ltd filed Critical Jilin Jihui Biotechnology Co Ltd
Priority to CN201810813395.5A priority Critical patent/CN108865989A/zh
Publication of CN108865989A publication Critical patent/CN108865989A/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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • C12N2500/20—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
    • C12N2500/30—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
    • C12N2500/38—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
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • C12N2500/76—Undefined extracts from plants
    • 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
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-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
    • C12N2501/10—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
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • 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
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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]
    • 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
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • C12N2501/33—Insulin
    • 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
    • C12N2501/335—Glucagon; Glucagon-like peptide [GLP]; Exendin
    • 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
    • C12N2501/73—Hydrolases (EC 3.)
    • 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

Abstract

一种脐带间充质干细胞的培养基，包括DMEM/F12培养基、添加在DMEM/F12培养基的添加成分和常春藤提取物，添加成分包括以下成分：人血清白蛋白、转铁蛋白、血管内皮生长因子、碱性成纤维细胞生长因子、精氨酸、白血病抑制因子、干细胞因子、β细胞素、甲状旁腺激素、血小板源性生长因子、肿瘤坏死因子α、白细胞介素、***、促血小板生成素、α‑D‑葡萄糖、维生素C、白藜芦醇、NaHCO3、神经节苷脂；常春藤提取物为液体，通过常规方法从常春藤的叶中提取，添加的终浓度为20～50mg/L。本发明解决了现有技术中存在的问题，使细胞能够保持良好的状态和增殖速度。

Description

一种脐带间充质干细胞的培养基
技术领域
本发明涉及干细胞培养技术领域，特别是涉及一种脐带间充质干细胞的培养基。
背景技术
脐带间充质干细胞是指存在于新生儿脐带组织中的一种多功能干细胞，它能分化成许多种组织细胞，具有广阔的临床应用前景。应用灭活脐带血清培养体系可成功扩增人脐带间充质干细胞，培养的细胞具有间充质干细胞的基本特性，为建立间充质干细胞库和临床应用提供了理论依据。间充质干细胞具有较强的免疫调节作用，能促进造血恢复功能，能修复损伤或病变的组织器官。目前，市场上非常多的间充质干细胞培养基产品，缺点：1、使用动物来源血清，同时也会引入病原体和异种抗原。美国FDA 正考虑禁止使用胎牛血清用于细胞治疗；2、干细胞容易分化，不能长期保持干性，且细胞的形态容易改变。
发明内容
本发明的目的在于提供一种脐带间充质干细胞的培养基，解决了现有技术中存在的动物来源血清会引入病原体和异种抗原的问题，并且使细胞能够保持良好的状态和增殖速度，培养基抗氧化效果好。
为实现上述目的，本发明是通过以下技术方案来实现的：
一种脐带间充质干细胞的培养基，包括DMEM/F12培养基、添加在所述DMEM/F12培养基的添加成分和常春藤提取物，所述添加成分包括以下终浓度的成分：人血清白蛋白1～5g/L、溶菌酶10mg/mL、转铁蛋白5～10mg/L、纤粘连蛋白2～8mg/L、层粘连蛋白1～4mg/L、转铁蛋白5～ 10mg/L、Fe(NO3)3·9H2O50g/L、FeSO4·7H2O417g/L、***1～3μg/L、睾酮2～5μg/L、***1～2μg/L、***39.25～117.74μg/L、胰岛素5～ 10mg/L、胰高血糖素样肽12μg/L、核黄素376.36mg/L、辅酶A80.96～ 242.87mg/L、丁二胺4.41～6.17mg/L、牛磺酸1～2mg/L、氨基乙醇0.61～ 1.85mg/L、丙酮酸8.81～26.42mg/L、硒酸钠3.78～7.56μg/L、血管内皮生长因子2～8μg/L、碱性成纤维细胞生长因子4～10μg/L、精氨酸10～ 20mg/L、白血病抑制因子1～5μg/L、干细胞因子2～8μg/L、β细胞素5～ 10μg/mL、甲状旁腺激素35.74～71.48ng/L、血小板源性生长因子AB0.5～ 2.5μg/L、肿瘤坏死因子α0.5～2.5μg/L、白细胞介素20.5～2.5μg/L、氢化可的松0.36～1.09mg/L、大豆胰酶抑制剂1～2mg/L、***1μg/L、促血小板生成素1μg/L、α-D-葡萄糖3135mg/L、维生素C 176.13mg/L、白藜芦醇4μg/L、NaHCO32.2g/L、神经节苷脂5μg/L；所述常春藤提取物为液体，通过常规方法从常春藤的叶中提取，添加的终浓度为20～50mg/L。将常春藤的叶片先进行粉碎，再发酵，预溶胀并在30％的乙醇溶液中进行提取，得到所述常春藤提取物。
优选地，所述添加成分还包括终浓度为1～4μg/L的转化生长因子α和/ 或1～4μg/L转化生长因子β。
优选地，所述添加成分还包括终浓度为1～2μg/L的花色苷。
本发明的有益效果，一是本发明不含动物血清，所有成分均来自人(如白蛋白、细胞因子等)，排除了动物血清内潜在动物源性内毒素或病毒，并且培养基中加入了溶菌酶，安全性高，方便应用于临床；二是本发明加入了常春藤提取物、花色苷，提高了抗氧化能力，促进细胞生长，有一定的修复效果。
附图说明
图1为本发明的实施例一培养出的脐带间充质干细胞形态照片。
图2为本发明的实施例一培养出的脐带间充质干细胞形态另一照片。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用组分、试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下实验中使用的组分、试剂和仪器如下：
下面对本发明作进一步说明如下：
脐带间充质干细胞的制备如下：
(1)洗涤脐带：无菌有齿镊转移脐带至10cm无菌培养皿中，加入标本洗涤液冲洗，重复2～3次，尽量去除血渍。
(2)无菌组织剪将脐带剪成约2～3cm数段，加入10～20ml标本洗涤液洗涤血凝块，重复洗涤直至基本去除血渍，洗涤液较清澈。剔除血管：按血管螺旋走势剔除脐带的两条动脉和一条静脉。
(3)将剔除血管的脐带组织用适量标本洗涤液洗涤血渍，重复洗涤直至洗涤液清澈。
(4)将洗涤后的脐带放入10cm无菌培养皿中，用无菌三头组织剪将脐带剪成组织均浆块。
(5)将组织均浆块平均接种至15cm培养皿中，组织块均匀分布，加入适量完全培养基。
(6)正置培养皿使组织块尽量均匀分布于整个底面，将培养皿放置于 CO2恒温恒湿培养箱中培养。培养条件：37±0.5℃，CO2体积分数为： 5.0±0.2％。
实施例一
一种脐带间充质干细胞的培养基，包括DMEM/F12培养基、添加在所述DMEM/F12培养基的添加成分和常春藤提取物，所述添加成分包括以下终浓度的成分：人血清白蛋白1.25g/L、溶菌酶10mg/mL、转铁蛋白7.5mg/L、纤粘连蛋白8mg/L、层粘连蛋白4mg/L、转铁蛋白10mg/L、Fe(NO3)3·9H2O 50g/L、FeSO4·7H2O 417g/L、***1.3μg/L、睾酮2μg/L、***1.3μg/L、***78.49μg/L、胰岛素7mg/L、胰高血糖素样肽12μg/L、核黄素 376.36mg/L、辅酶A161.91mg/L、丁二胺5.30mg/L、牛磺酸1.25mg/L、氨基乙醇1.22mg/L、丙酮酸22.42mg/L、硒酸钠5.67μg/L、血管内皮生长因子6μg/L、碱性成纤维细胞生长因子10μg/L、精氨酸20mg/L、白血病抑制因子1μg/L、干细胞因子8μg/L、β细胞素10μg/mL、甲状旁腺激素35.74ng/L、血小板源性生长因子AB2.2μg/L、肿瘤坏死因子α2.5μg/L、白细胞介素2 2.5μg/L、氢化可的松0.36mg/L、大豆胰酶抑制剂1mg/L、*** 1μg/L、促血小板生成素1μg/L、α-D-葡萄糖3135mg/L、维生素C 176.13mg/L、白藜芦醇4μg/L、NaHCO32.2g/L、神经节苷脂5μg/L、转化生长因子α2μg/L、花色苷2μg/L；所述常春藤提取物为液体，通过常规方法从常春藤的叶中提取，添加的终浓度为50mg/L。
将各成分按其各自特性进行溶解，混合，HEPES定容，0.22μm过滤膜过滤除菌，得到培养基Ⅰ，4℃保存。
实施例二
一种脐带间充质干细胞的培养基，包括DMEM/F12培养基、添加在所述DMEM/F12培养基的添加成分和常春藤提取物，所述添加成分包括以下终浓度的成分：人血清白蛋白5g/L、溶菌酶10mg/mL、转铁蛋白5mg/L、纤粘连蛋白2mg/L、层粘连蛋白4mg/L、转铁蛋白5mg/L、Fe(NO3)3·9H2O 50g/L、FeSO4·7H2O 417g/L、***3μg/L、睾酮5μg/L、***1μg/L、***39.25μg/L、胰岛素10mg/L、胰高血糖素样肽12μg/L、核黄素 376.36mg/L、辅酶A161.91mg/L、丁二胺5.30mg/L、牛磺酸1.25mg/L、氨基乙醇0.61mg/L、丙酮酸22.42mg/L、硒酸钠3.78μg/L、血管内皮生长因子2μg/L、碱性成纤维细胞生长因子4μg/L、精氨酸10mg/L、白血病抑制因子1μg/L、干细胞因子8μg/L、β细胞素5μg/mL、甲状旁腺激素71.48ng/L、血小板源性生长因子AB0.5μg/L、肿瘤坏死因子α0.5μg/L、白细胞介素2 0.5μg/L、氢化可的松1.09mg/L、大豆胰酶抑制剂2mg/L、*** 1μg/L、促血小板生成素1μg/L、α-D-葡萄糖3135mg/L、维生素C 176.13mg/L、白藜芦醇4μg/L、NaHCO32.2g/L、神经节苷脂5μg/L、转化生长因子β2μg/L、花色苷2μg/L；所述常春藤提取物为液体，通过常规方法从常春藤的叶中提取，添加的终浓度为20mg/L。
将各成分按其各自特性进行溶解，混合，HEPES定容，0.22μm过滤膜过滤除菌，得到培养基Ⅱ，4℃保存。
对比例一
间充质干细胞生长无血清培养基，来源LONZA，货号00190632，标记为培养基Ⅲ，4℃保存。
细胞增值速度对比实验
取生长状态良好的脐带间充质干细胞，用0.05％胰酶-EDTA消化细胞，消化至在倒置显微镜下观察到细胞大部分由梭形变为圆形并脱落，加入培养基Ⅰ、培养基Ⅱ和培养基Ⅲ，按5×104/cm2接种于96孔培养板中，每孔分别加上述两培养基100μl。同时取培养板一列加入生长培养基但不加细胞，每孔培养基为100μl，作为空白对照组。每12h取4孔，MTT比色法测570nm波长光吸收值(D570值)，连续测3天，实验得到的结果如表1、表2、表3所示。
表1脐带间充质干细胞在培养基Ⅰ培养增值速度表
时间 12h 24h 36h 48h 60h 72h 84h
D570值 0.12 0.25 0.39 0.4 0.4 0.4 0.4
表2脐带间充质干细胞在培养基Ⅱ培养增值速度表
时间 12h 24h 36h 48h 60h 72h 84h
D570值 0.11 0.22 0.35 0.4 0.4 0.4 0.4
表3脐带间充质干细胞在培养基Ⅲ培养增值速度表
时间 12h 24h 36h 48h 60h 72h 84h
D570值 0.08 0.12 0.22 0.32 0.4 0.4 0.4
培养基Ⅰ和培养基Ⅱ均采用本发明的组分和方法配制，区别在于添加成分的终浓度不同。培养基Ⅲ为市面上使用较多的间充质干细胞生长无血清培养基。从表1、表2、表3可以得出，采用本发明的培养基Ⅰ和培养基Ⅱ培养的脐带间充质干细胞，细胞增值速度均明显优于培养基Ⅲ培养的脐带间充质干细胞。
实施例一的培养基Ⅰ培养的干细胞均呈良好状态，如图1和图2所示。
本发明不含动物血清，所有成分均来自人(如白蛋白、细胞因子等)，排除了动物血清内潜在动物源性内毒素或病毒，并且培养基中加入了溶菌酶，安全性高，方便应用于临床。本发明加入了常春藤提取物、白藜芦醇、花色苷，提高了抗氧化能力，促进细胞生长，有一定的修复效果，培养出的细胞形态良好。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用。它完全可以被适用于各种适合本发明的领域。对于熟悉本领域的人员而言，可容易地实现另外的修改。因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图。

Claims (3)

1.一种脐带间充质干细胞的培养基，其特征在于，包括DMEM/F12培养基、添加在所述DMEM/F12培养基的添加成分和常春藤提取物，所述添加成分包括以下终浓度的成分：人血清白蛋白1～5g/L、溶菌酶10mg/mL、转铁蛋白5～10mg/L、纤粘连蛋白2～8mg/L、层粘连蛋白1～4mg/L、转铁蛋白5～10mg/L、Fe(NO3)3·9H2O50g/L、FeSO4·7H2O417g/L、***1～3μg/L、睾酮2～5μg/L、***1～2μg/L、***39.25～117.74μg/L、胰岛素5～10mg/L、胰高血糖素样肽12μg/L、核黄素376.36mg/L、辅酶A80.96～242.87mg/L、丁二胺4.41～6.17mg/L、牛磺酸1～2mg/L、氨基乙醇0.61～1.85mg/L、丙酮酸8.81～26.42mg/L、硒酸钠3.78～7.56μg/L、血管内皮生长因子2～8μg/L、碱性成纤维细胞生长因子4～10μg/L、精氨酸10～20mg/L、白血病抑制因子1～5μg/L、干细胞因子2～8μg/L、β细胞素5～10μg/mL、甲状旁腺激素35.74～71.48ng/L、血小板源性生长因子AB0.5～2.5μg/L、肿瘤坏死因子α0.5～2.5μg/L、白细胞介素20.5～2.5μg/L、氢化可的松0.36～1.09mg/L、大豆胰酶抑制剂1～2mg/L、***1μg/L、促血小板生成素1μg/L、α-D-葡萄糖3135mg/L、维生素C 176.13mg/L、白藜芦醇4μg/L、NaHCO32.2g/L、神经节苷脂5μg/L；所述常春藤提取物为液体，通过常规方法从常春藤的叶中提取，添加的终浓度为20～50mg/L。
2.根据权利要求1所述的一种脐带间充质干细胞的培养基，其特征在于，所述添加成分还包括终浓度为1～4μg/L的转化生长因子α和/或1～4μg/L转化生长因子β。
3.根据权利要求1所述的一种脐带间充质干细胞的培养基，其特征在于，所述添加成分还包括终浓度为1～2μg/L的花色苷。
CN201810813395.5A 2018-07-23 2018-07-23 一种脐带间充质干细胞的培养基 Pending CN108865989A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810813395.5A CN108865989A (zh) 2018-07-23 2018-07-23 一种脐带间充质干细胞的培养基

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810813395.5A CN108865989A (zh) 2018-07-23 2018-07-23 一种脐带间充质干细胞的培养基

Publications (1)

Publication Number Publication Date
CN108865989A true CN108865989A (zh) 2018-11-23

Family

ID=64304249

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810813395.5A Pending CN108865989A (zh) 2018-07-23 2018-07-23 一种脐带间充质干细胞的培养基

Country Status (1)

Country Link
CN (1) CN108865989A (zh)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317779A (zh) * 2019-06-06 2019-10-11 深圳市艾一生命科技有限公司 无血清的脐带间充质干细胞培养基
CN110331130A (zh) * 2019-07-03 2019-10-15 依科赛生物科技（太仓）有限公司 一种间充质干细胞无血清培养基及其用途
CN111518761A (zh) * 2020-05-25 2020-08-11 青岛瑞思德生物科技有限公司 一种间充质干细胞体外筛选、激活、扩增、冻存及其细胞库建立的方法
CN114591901A (zh) * 2022-03-24 2022-06-07 和携科技有限公司 动物脐带间充质干细胞外泌体提取方法
CN116410921A (zh) * 2023-02-09 2023-07-11 北京益华生物科技有限公司 一种人源脐带间充质干细胞诱导培养基、诱导方法及应用
EP4092106A4 (en) * 2020-01-13 2024-03-06 Qingdao Restore Biotechnology Co Ltd INDUCER FOR INDUCING MESENCHYMAL STEM CELLS TO DIFFERENTIATE INTO ISLAND CELLS

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101363012A (zh) * 2008-09-27 2009-02-11 上海交通大学 以rhDSL重组蛋白扩增造血干细胞及祖细胞的方法
CN103243071A (zh) * 2013-05-09 2013-08-14 陈云燕 一种临床级人间充质干细胞无血清完全培养基
CN104164405A (zh) * 2014-08-12 2014-11-26 赛业（苏州）生物科技有限公司 一种高效的体外培养人脐带间质干细胞的无血清培养体系
CN105132369A (zh) * 2015-08-25 2015-12-09 北京瑞思德生物医学研究院 一种将间充质干细胞转化为睾酮分泌细胞的诱导剂及诱导培养基
CN106635978A (zh) * 2016-12-21 2017-05-10 广东科玮生物技术股份有限公司 一种脐带间充质干细胞无血清培养基及其制备方法和用途
CN107043747A (zh) * 2017-06-23 2017-08-15 王晓柯 一种人脐带间充质干细胞无血清培养基

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101363012A (zh) * 2008-09-27 2009-02-11 上海交通大学 以rhDSL重组蛋白扩增造血干细胞及祖细胞的方法
CN103243071A (zh) * 2013-05-09 2013-08-14 陈云燕 一种临床级人间充质干细胞无血清完全培养基
CN104164405A (zh) * 2014-08-12 2014-11-26 赛业（苏州）生物科技有限公司 一种高效的体外培养人脐带间质干细胞的无血清培养体系
CN105132369A (zh) * 2015-08-25 2015-12-09 北京瑞思德生物医学研究院 一种将间充质干细胞转化为睾酮分泌细胞的诱导剂及诱导培养基
CN106635978A (zh) * 2016-12-21 2017-05-10 广东科玮生物技术股份有限公司 一种脐带间充质干细胞无血清培养基及其制备方法和用途
CN107043747A (zh) * 2017-06-23 2017-08-15 王晓柯 一种人脐带间充质干细胞无血清培养基

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
周婷婷等: "无血清培养体系原代培养脐带间充质干细胞", 《中国组织工程研究》 *
孙亚如等: "培养体系对维持人脐带间充质干细胞特性及其体外扩增效率的影响", 《中国组织工程研究》 *
王振宇等: "《天然产物分离技术》", 31 January 2012, 北京：中国轻工业出版社 *
赵中振等: "《当代药用植物典 第3册》", 29 February 2008, 世界图书出版公司 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317779A (zh) * 2019-06-06 2019-10-11 深圳市艾一生命科技有限公司 无血清的脐带间充质干细胞培养基
CN110331130A (zh) * 2019-07-03 2019-10-15 依科赛生物科技（太仓）有限公司 一种间充质干细胞无血清培养基及其用途
EP4092106A4 (en) * 2020-01-13 2024-03-06 Qingdao Restore Biotechnology Co Ltd INDUCER FOR INDUCING MESENCHYMAL STEM CELLS TO DIFFERENTIATE INTO ISLAND CELLS
CN111518761A (zh) * 2020-05-25 2020-08-11 青岛瑞思德生物科技有限公司 一种间充质干细胞体外筛选、激活、扩增、冻存及其细胞库建立的方法
CN111518761B (zh) * 2020-05-25 2022-04-29 青岛瑞思德生物科技有限公司 一种间充质干细胞体外筛选、激活、扩增、冻存及其细胞库建立的方法
CN114591901A (zh) * 2022-03-24 2022-06-07 和携科技有限公司 动物脐带间充质干细胞外泌体提取方法
CN114591901B (zh) * 2022-03-24 2023-11-24 和携科技有限公司 动物脐带间充质干细胞外泌体提取方法
CN116410921A (zh) * 2023-02-09 2023-07-11 北京益华生物科技有限公司 一种人源脐带间充质干细胞诱导培养基、诱导方法及应用
CN116410921B (zh) * 2023-02-09 2024-01-23 北京益华生物科技有限公司 一种人源脐带间充质干细胞诱导培养基、诱导方法及应用

Similar Documents

Publication Publication Date Title
CN108865989A (zh) 一种脐带间充质干细胞的培养基
CN103404509B (zh) 一种细胞保存液、其制备方法和应用
CN104805054B (zh) 干细胞无血清培养基
Shiota et al. Development of the fetal mouse palate in suspension organ culture
CN106176563B (zh) 人脐带msc无血清培养液脂质体冻干粉及其制备和应用
CN110564682B (zh) 一种大规模生产人脂肪间充质干细胞外泌体的方法
CN101412985A (zh) 用于骨髓间充质干细胞体外培养和扩增的无血清培养基
CN108823156A (zh) 用于修复的临床级人脐带间充质干细胞复合因子及冻干粉的制备方法
CN102807965B (zh) 一种组织工程角膜的制备方法及其装置
CN103893825B (zh) 含胶原蛋白的细菌纤维素复合羊膜细胞外基质材料制备方法
CN106754639A (zh) 一种间充质干细胞因子大规模制备方法
CN101757691A (zh) 一种组织工程角膜的制备方法
CN105670986A (zh) 一种诱导人脐带间充质干细胞分化为胰岛样细胞的培养基及其诱导方法
CN109022343A (zh) 一种人参干细胞的制备方法
CN106038598A (zh) 人源干细胞分泌生物活性因子与裂解液的制备方法
CN107129967A (zh) 一种用于原代人表皮细胞培养的无血清培养基体系
CN110305839A (zh) 间充质干细胞无血清培养基
CN107418928A (zh) 一种人间充质干细胞成骨诱导分化培养基及制备方法
CN107362391A (zh) 一种自体三联皮肤成纤维细胞制剂的制备方法
CN111557893A (zh) 一种含有干细胞外泌体成分的面膜制备方法
CN102660496A (zh) 包含来自Cohn分级分离阶段上清液的哺乳动物细胞培养基及其用途
WO2021098025A1 (zh) 一种体外激活脂肪干细胞转化成原软骨细胞的方法
CN112655554B (zh) 一种基于led光质加速诱导苹果愈伤组织及生长的方法
CN109385397A (zh) 一种培养间充质干细胞的无血清培养基及其制备方法
CN107129966A (zh) 一种含血清的角膜上皮细胞培养液

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
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20200515

Address after: 266000, No. 1, 2, building 7, Qingdao blue bio Pharmaceutical Industrial Park, 368 Hedong Road, Hedong District, Qingdao, Shandong

Applicant after: QINGDAO RE-STORE BIOTECHNOLOGY Co.,Ltd.

Address before: 130000 North Lake Science and Technology Development Zone, Changchun, Jilin, China, 501 A11, North Lake Science and Technology Park, 3333 Shengbei street.

Applicant before: JILIN JIHUI BIOTECHNOLOGY Co.,Ltd.

WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20181123