CN108130310A - 一种干细胞培养基 - Google Patents

一种干细胞培养基 Download PDF

Info

Publication number
CN108130310A
CN108130310A CN201611077742.XA CN201611077742A CN108130310A CN 108130310 A CN108130310 A CN 108130310A CN 201611077742 A CN201611077742 A CN 201611077742A CN 108130310 A CN108130310 A CN 108130310A
Authority
CN
China
Prior art keywords
stem cell
cell media
cell
iron
food additives
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
CN201611077742.XA
Other languages
English (en)
Inventor
陈文杰
陈巧云
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.)
Nanjing ruikangjian Biomedical Technology Co.,Ltd.
Original Assignee
JIANGYIN RUIKANGJIAN BIOMEDICAL SCIENCE TECHNOLOGY 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 JIANGYIN RUIKANGJIAN BIOMEDICAL SCIENCE TECHNOLOGY CO LTD filed Critical JIANGYIN RUIKANGJIAN BIOMEDICAL SCIENCE TECHNOLOGY CO LTD
Priority to CN201611077742.XA priority Critical patent/CN108130310A/zh
Publication of CN108130310A publication Critical patent/CN108130310A/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
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • 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
    • 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
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • 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
    • C12N2501/395—Thyroid hormones

Abstract

本发明涉及一种干细胞培养基，包括基础培养基，细胞生长因子、谷氨酰胺、含铁的食品添加剂。本发明基能够快速扩增干细胞同时又不影响干细胞的潜能，干细胞的扩增速度较常规培养基提高3‑5倍，而且能用于培养多种组织的干细胞，具有极佳的适用性，所培养的干细胞分化能力强，可分化成多种功能细胞，具有很高的科研和医学应用价值。

Description

一种干细胞培养基
技术领域
本发明在于干细胞培养基及方法领域。
背景技术
诸多研究表明干细胞不仅可以自我更新，在条件合适的情况下能分化成其他功能细胞，因此干细胞有望成为治疗人类疑难疾病的有效手段。然而，干细胞在正常成体组织中含量甚微，在体外如何快速扩增与培养干细胞培养是研究干细胞的作用机制及探索其在治疗人类疾病治疗方法的重要技术。
干细胞尽量含量甚微，但广泛分布于哺乳动物的各个组织器官，这些组织或器官包括但不局限于骨髓、脐带、脂肪组织、脑组织、视网膜、心脏、肝脏、肺以及皮肤等。
目前，在干细胞的常规培养体系中均加入了一定比例的动物血清，比较常用的是胎牛血清或新生小牛血清。血清是由很多大小不同生物分子组成的极为复杂的混合物，它对细胞在体外培养时的主要作用是提供生长因子、激素、结合蛋白，并提供保护作用。
发明内容
本发明提供的干细胞培养基，该培养基能够快速扩增干细胞同时又不影响干细胞的潜能，干细胞的扩增速度较常规培养基提高3-5倍，而且能用于培养多种组织的干细胞，具有极佳的适用性，所培养的干细胞分化能力强，可分化成多种功能细胞，具有很高的科研和医学应用价值。
本发明的技术方案如下：
一种干细胞培养基，其中，所述培养基组分包括基础培养基，细胞因子、谷氨酰胺、含铁的食品添加剂；
所述基础培养基为DMEM/F12培养基；
所述的细胞因子为甲状腺素、肾上腺素和胰岛素中一种或者多种；
所述含铁的食品添加剂包括转铁蛋白、柠檬酸铁和葡糖酸铁中一种或多种。
在本发明提供的干细胞培养基中，所述干细胞培养基中，细胞因子的浓度为1-15μg/ml、谷氨酰胺的浓度为1-5mmol/l、含铁的食品添加剂的浓度为20-200ng/ml。
在本发明提供的干细胞培养基中，还包括HEPES、青霉素和链霉素。
在本发明提供的干细胞培养基中，所述HEPES的浓度为2-20mmol/L，青霉素的浓度为1-10mg/L，链霉素的浓度为1-10mg/L。
附图说明
图1为本发明的培养基对人尿源干细胞增殖的加速作用的实验结果。
具体实施方式
实施例1：
对干细胞增殖的加速作用的实验
1.受试培养基：常规培养基组份：DMEM/F12基本培养基，10％胎牛血清(FBS)；本发明的培养基，包括：DMEM/F12基础培养基、甲状腺素50ng/ml、肾上腺素500ng/ml、胰岛素25ug/ml、转铁蛋白20ug/ml、谷氨酰胺5mM/L、HEPES 25mM/L、青霉素1mg/L和链霉素1mg/L。
2.培养干细胞来源：来源于人尿的尿源干细胞
3.体外细胞培养实验：
在无菌条件下，取健康志愿者清洁中段尿液200-250ml，为防止感染，加入含有青霉素100U/ml加链霉素0.1mg/ml的双抗5ml，并立即分离提取。将清洁尿液分装至50ml离心管中，400g离心10min，弃上清。经PBS以400g离心10min洗涤两次。取100μL细胞重悬液并应用台盼蓝检测细胞活性并计数，调整细胞密度，接种至含有无血清培养基的六孔板中，置于37℃C下5％CO2饱和湿度的孵箱中培养。14-21天后，可以陆续观察到克隆逐渐向四周延伸，克隆逐渐变大。待细胞达到80％左右的融合度后，胰蛋白酶消化离心，重悬后得到尿液间充质干细胞。
干细胞接种密度为4000个/ml，接种于12孔板中放入培养箱培养(37℃，5％CO2)。每隔24小时里利用血球计数板测一次细胞数量，结果发现，经本发明的培养基培养的尿源干细胞比常规培养基培养的细胞便显出更快的扩增速度(数据为三次独立实验的平均值)。
实施例2：
对增殖后的尿源干细胞体外分化为成软骨细胞的实验
1.受试培养基：本发明的培养基。
2.培养细胞来源：人尿来源的尿源干细胞
3.尿液间充质干细胞向软骨细胞的分化：
(1)取实施案例1所获得的尿液间充质干细胞第四代培养
(2)向尿液间充质干细胞中加入含有转化生长因子β110ng/ml、胰岛素样生长因子100ng/ml和地塞米松0.1μmol/L的诱导培养基
(3)诱导培养28天后，阿尔新蓝染色细胞细胞外基质，免疫组化检测II型胶原表达情况。
结果显示，诱导培养后的尿液间充质干细胞大部分阿尔新蓝着色，II型胶原表达增强，表明尿液间充质干细胞可以向软骨细胞分化。

Claims (5)

1.一种干细胞培养基，包括基础培养基，其特征在于：还包括细胞生长因子、谷氨酰胺、含铁的食品添加剂。
2.根据权利要求1所述的干细胞培养基，其特征在于，所述细胞生长因子包括甲状腺素、肾上腺素、胰岛素中的一种或者多种。
3.根据权利要求1所述的干细胞培养基，其特征在于，所述含铁的食品添加剂包括转铁蛋白、柠檬酸铁和葡糖酸铁中的一种或多种。
4.根据权利要求1所述的干细胞培养基，其特征在于，所述干细胞培养基中细胞因子的浓度为1-15μg/ml、谷氨酰胺的浓度为1-5mmol/L、含铁的食品添加剂的浓度为20-200ng/ml。
5.根据权利要求1所述的干细胞培养基，其特征在于，所述基础培养基为DMEM/F12基础培养基。
CN201611077742.XA 2016-11-30 2016-11-30 一种干细胞培养基 Pending CN108130310A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611077742.XA CN108130310A (zh) 2016-11-30 2016-11-30 一种干细胞培养基

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611077742.XA CN108130310A (zh) 2016-11-30 2016-11-30 一种干细胞培养基

Publications (1)

Publication Number Publication Date
CN108130310A true CN108130310A (zh) 2018-06-08

Family

ID=62387928

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611077742.XA Pending CN108130310A (zh) 2016-11-30 2016-11-30 一种干细胞培养基

Country Status (1)

Country Link
CN (1) CN108130310A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115369080A (zh) * 2022-08-08 2022-11-22 北京大学人民医院 一种临床级自体尿源干细胞培养基及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010095684A1 (ja) * 2009-02-23 2010-08-26 富士レビオ株式会社 脂肪細胞への分化誘導培地及び方法
JP2012210201A (ja) * 2011-03-18 2012-11-01 Otsuka Pharmaceut Factory Inc 間葉系幹細胞の増殖促進剤
CN102925409A (zh) * 2012-11-19 2013-02-13 上海市第六人民医院 尿液间充质干细胞的提取及扩增培养方法和应用
CN103060264A (zh) * 2012-12-20 2013-04-24 上海市第十人民医院 一种干细胞培养基及其应用和干细胞培养方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010095684A1 (ja) * 2009-02-23 2010-08-26 富士レビオ株式会社 脂肪細胞への分化誘導培地及び方法
JP2012210201A (ja) * 2011-03-18 2012-11-01 Otsuka Pharmaceut Factory Inc 間葉系幹細胞の増殖促進剤
CN102925409A (zh) * 2012-11-19 2013-02-13 上海市第六人民医院 尿液间充质干细胞的提取及扩增培养方法和应用
CN103060264A (zh) * 2012-12-20 2013-04-24 上海市第十人民医院 一种干细胞培养基及其应用和干细胞培养方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JUN-JIE GUAN: "Biological Characteristics of Human-Urine-Derived Stem Cells: Potential for Cell-Based Therapy in Neurology", 《TISSUE ENG PART A》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115369080A (zh) * 2022-08-08 2022-11-22 北京大学人民医院 一种临床级自体尿源干细胞培养基及其应用

Similar Documents

Publication Publication Date Title
CN101460610B (zh) 修复和/或再生受损心肌的方法和组合物
Danoviz et al. Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system
Kreuz et al. Platelet-rich plasma preparation types show impact on chondrogenic differentiation, migration, and proliferation of human subchondral mesenchymal progenitor cells
Baglioni et al. Functional differences in visceral and subcutaneous fat pads originate from differences in the adipose stem cell
Ehrhardt et al. Regenerative capacity of skeletal muscle
CN101748096A (zh) 亚全能干细胞、其制备方法及其用途
Grasys et al. Content of soluble factors and characteristics of stromal vascular fraction cells in lipoaspirates from different subcutaneous adipose tissue depots
Bellio et al. Physiological and hypoxic oxygen concentration differentially regulates human c-Kit+ cardiac stem cell proliferation and migration
Peppicelli et al. Anoikis resistance as a further trait of acidic-adapted melanoma cells
KR20080042761A (ko) 렙틴을 이용한 줄기세포 증식방법
JP2023060125A (ja) 表皮水疱症の治療剤
Yang et al. Platelet poor plasma gel combined with amnion improves the therapeutic effects of human umbilical cord‑derived mesenchymal stem cells on wound healing in rats
EP1637589A1 (en) Novel stem cell derived from adult tissue and use thereof
Tamaki et al. Clonal Differentiation of Skeletal Muscle–Derived CD34−/45− Stem Cells Into Cardiomyocytes In Vivo
CN102282250A (zh) 使哺乳动物祖细胞分化为产生胰岛素的胰岛细胞的方法
CN102712897B (zh) 心脏组织来源细胞
CN108130310A (zh) 一种干细胞培养基
CN108570446A (zh) 一种尿液来源细胞的培养方法
Popuri Concerns of a pediatric dentist in dental stem cells: an overview
CN102703380B (zh) 亚全能干细胞、其制备方法及其用途
Hong et al. Natural killer cells inhibit pulmonary metastasis of hepatocellular carcinoma in nude mice
Yap et al. Pluripotent stem cell-derived committed cardiac progenitors remuscularize damaged ischemic hearts and improve their function in pigs
Siddiqui et al. Modulatory effects of cell–cell interactions between porcine skeletal muscle satellite cells and fibroblasts on the expression of myogenesis-related genes
US20120201786A1 (en) Methods for the use of stem cells and stem cell factors in the treatment of skin conditions
Li et al. Are bone marrow regenerative cells ideal seed cells for the treatment of cerebral ischemia?★

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: 20210608

Address after: Room 923-2, building a, phase I, Zhongdan Ecological Life Science Industrial Park, no.3-1, xinjinhu Road, Jiangbei new district, Nanjing City, Jiangsu Province, 210000

Applicant after: Nanjing ruikangjian Biomedical Technology Co.,Ltd.

Address before: 214437 2nd floor, building D2, No.6 Dongsheng West Road, Chengdong street, Wuxi City, Jiangsu Province

Applicant before: Jiangyin Recongene Biomedical Technologies Co.,Ltd.

DD01 Delivery of document by public notice
DD01 Delivery of document by public notice

Addressee: Chen Qiaoyun

Document name: Notification of eligibility

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

Application publication date: 20180608