CN109337866A - 一种脂肪干细胞无血清培养基 - Google Patents
一种脂肪干细胞无血清培养基 Download PDFInfo
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Abstract
本发明公开了一种人脂肪干细胞无血清培养基,主要成分包括氨基酸,无机盐成分,生长因子及白蛋白成分。本发明提供了一种人脂肪干细胞无血清培养基,其不含动物源成分,可以完全实现无血清培养干细胞。采用本发明的无血清培养基培养人脂肪干细胞,可在体外获得高数量、高纯度并且安全优质的间充质干细胞,同时可提高细胞存活率,维持干细胞的特性。本发明可解决细胞数量不够的问题,且每批次的细胞数量、存活率及免疫表型更加稳定。
Description
技术领域
本发明涉及一种人脂肪干细胞无血清培养基,属于细胞工程技术领域。
背景技术
间质干细胞(mesenchymal stem cells,MSCs)来源于中胚层,也称多能间充质基质细胞(multipotent mesenchymal stromal cells),为多能干细胞的一种,它最早发现于骨髓中,具有向三种胚层细胞分化的多向分化潜能,具有低免疫原性、不刺激产生同种异体免疫反应、不被细胞毒性T细胞和NK细胞杀伤,以及免疫调节作用。MSCs在相应的诱导条件下能分化为骨、脂肪、软骨、肌肉和肝细胞等不同类型细胞。脂肪干细胞(adipose derivedstem cslls,ADSC)是一种存在于脂肪组织中的成体间充质干细胞(mesenchymal stemcells,MSC)。在特定诱导条件下可以分化为不同胚层细胞和组织。2001年脂肪干细胞首次通过吸脂手术并从人脂肪组织悬液中分离获得。由于人脂肪组织含量丰富且容易获得,所以脂肪干细胞是成体干细胞的又一丰富补充来源。此外,脂肪干细胞不表达 II 类主要组织相容性复合体,表明脂肪干细胞具有低免疫原性的特点,适合同种异体移植。基于以上获取简便、来源广泛、高分化潜能和低免疫原性等优势,脂肪干细胞被广泛应用于细胞工程、组织工程、医学等各个领域,并有着广阔的发展前景。
细胞培养基是决定细胞体外生长代谢最重要、最直接的环境因素。目前大多数合成细胞培养基均需要补充动物血清才能支持细胞的体外生长、增殖。传统含血清培养基成分复杂且不明确,无法证明其安全性,批次之间差异较大,无法满足干细胞相关药物及干细胞医疗的开发要求。另外,市售无血清培养基不仅价格昂贵,而且对于脂肪干细胞培养针对性不强,故而造成培养效果并不理想,脂肪干细胞生长缓慢且传代不稳定。为解决现存问题,我们针对于脂肪干细胞特点开发出了一种脂肪干细胞无血清培养基,保留无血清培养基优点的同时,更适合对脂肪干细胞进行培养。
发明内容
针对上述现有技术,本发明提供了一种人脂肪干细胞无血清培养基,其不含动物源成分,可以完全实现无血清培养干细胞。采用本发明的无血清培养基培养人脂肪干细胞,可在体外获得高数量、高纯度并且安全优质的间充质干细胞,同时可提高细胞存活率,维持干细胞的特性。本发明可解决细胞数量不够的问题,且每批次的细胞数量、存活率及免疫表型更加稳定。
本发明是通过以下技术方案实现的:
一种人脂肪干细胞无血清培养基,是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸 105~305;
CuSO4·5H2O 0.0006~0.005;
L-天门冬酰胺28~70;
L-天门冬氨酸12~32;
L-谷氨酸 15~35;
Ni(NO3)2·6H2O 0.000032~0.0003;
L-谷氨酰胺0~500;
ZnSO4·7H2O 0.065~0.65;
甘氨酸 5~20;
CoCl2·6H2O 0.001~0.008;
L-组氨酸5~35;
NaSiO3·9H2O 0.001~0.01;
L-异亮氨酸 20~75;
Na3VO4·12H2O 0.0005~0.005;
L-赖氨酸盐酸25~50;
SnCl2·2H2O 0.00001~0.0001;
L-蛋氨酸15~35;
Na2SeO3 0.002~0.01;
L-苯丙氨酸5~25;
FeSO4·7H2O 0.1~1.5;
L-脯氨酸5~30;
葡萄糖 1000~4000;
L-丝氨酸10~40;
维生素C 0.19~0.7;
L-苏氨酸10~30;
对羟基苯甲酸0.3~1.8;
L-色氨酸5~20;
丙酮酸钠 40~500;
L-缬氨酸15~30;
亚油酸 0.015~0.04;
L-亮氨酸 20~65;
β-巯基乙醇 0.5~3.5;
二水L-酪氨酸二钠120~420;
乙醇胺0.5~5;
L-胱氨酸二盐酸20~65;
地塞米松 0.0015~0.0045;
人转铁蛋白 5~45;
人血清白蛋白 1000~5500;
纤粘连蛋白 0.5~5;
重组人成纤维细胞生长因子 0.1~15;
重组人转化因子β 0.1~15;
重组人表皮生长因子0.1~15;
胰岛素:5~20;
纯化人转铁蛋白液:1~25;
胆固醇:15~55;
过氧化氢酶:15~45;
亚硒酸钠:15.3~35.5;
1%(质量百分数)二巯基乙醇溶液:0.25~1.5ml/L;
余量为水。
所述重组人成纤维细胞生长因子、重组人转化因子、重组人表皮生长因子均可市场购买得到,本发明所用为进口产品,均购自peprotech,商品名称为:EGF(重组人表皮生长因子),商品号为AF-100-15-50;商品名称为TGF-b(重组人转化因子),货号:100-21-100;商品名为(FGF)成纤维细胞生长因子,商品货号:100-18B-50。
本发明的无血清培养基的制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH 值至7.0~7.4,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
本发明的人脂肪干细胞无血清培养基,不含任何血清组分,且成分明确,避免了使用外源血清等成分不明物质带来的潜藏隐患,解决了应用的安全性问题。
采用本发明的无血清培养基培养人脂肪干细胞,细胞扩增倍数高、细胞形态更加均匀、饱满,表型稳定,干性维持的很好,符合相关指标要求。
具体实施方式
下面结合实施例对本发明作进一步的说明。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1 制备人脂肪干细胞无血清培养基
各原料的浓度如下,各浓度单位若无特别说明,均为mg/L:
L-精氨酸 210;
CuSO4·5H2O 0.002;
L-天门冬酰胺55;
L-天门冬氨酸20;
L-谷氨酸 25;
Ni(NO3)2·6H2O 0.00012;
L-谷氨酰胺300;
ZnSO4·7H2O 0.32;
甘氨酸 12;
CoCl2·6H2O 0.0055;
L-组氨酸15;
NaSiO3·9H2O 0.006;
L-异亮氨酸 45;
Na3VO4·12H2O 0.0015;
L-赖氨酸盐酸35;
SnCl2·2H2O 0.000045;
L-蛋氨酸20;
Na2SeO3 0.006;
L-苯丙氨酸18;
FeSO4·7H2O 1.0;
L-脯氨酸20;
葡萄糖 3000;
L-丝氨酸25;
维生素C 0.45;
L-苏氨酸25;
对羟基苯甲酸1.0;
L-色氨酸15;
丙酮酸钠 250;
L-缬氨酸20;
亚油酸 0.025;
L-亮氨酸 45;
β-巯基乙醇 2.2;
二水L-酪氨酸二钠280;
乙醇胺 2.5;
L-胱氨酸二盐酸45;
地塞米松 0.002;
人转铁蛋白 30;
人血清白蛋白 2200;
纤粘连蛋白 2.5;
重组人成纤维细胞生长因子 5;
重组人转化因子β 5;
重组人表皮生长因子5;
胰岛素:10;
纯化人转铁蛋白:20;
胆固醇:30;
过氧化氢酶:25 ;
亚硒酸钠:20.5;
1%二巯基乙醇溶液:0.65ml/L;
余量为水。
制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH 值至7.0~7.4,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
实施例2 制备人脂肪干细胞无血清培养基
各原料的浓度如下,各浓度单位若无特别说明,均为mg/L:
L-精氨酸 105;
CuSO4·5H2O 0.0006;
L-天门冬酰胺70;
L-天门冬氨酸32;
L-谷氨酸 15;
Ni(NO3)2·6H2O 0.000032;
L-谷氨酰胺500;
ZnSO4·7H2O 0.65;
甘氨酸 5;
CoCl2·6H2O 0.001;
L-组氨酸35;
NaSiO3·9H2O 0.01;
L-异亮氨酸 20;
Na3VO4·12H2O 0.0005;
L-赖氨酸盐酸50;
SnCl2·2H2O 0.0001;
L-蛋氨酸15;
Na2SeO3 0.002;
L-苯丙氨酸25;
FeSO4·7H2O 1.5;
L-脯氨酸5;
葡萄糖 1000;
L-丝氨酸40;
维生素C 0.7;
L-苏氨酸10;
对羟基苯甲酸0.3;
L-色氨酸20;
丙酮酸钠 500;
L-缬氨酸15;
亚油酸 0.015;
L-亮氨酸 65;
β-巯基乙醇 3.5;
二水L-酪氨酸二钠120;
乙醇胺 0.5;
L-胱氨酸二盐酸65;
地塞米松 0.0045;
人转铁蛋白 5;
人血清白蛋白 1000;
纤粘连蛋白 5;
重组人成纤维细胞生长因子 15;
重组人转化因子β 0.1;
重组人表皮生长因子0.1;
胰岛素: 20;
纯化人转铁蛋白: 25;
胆固醇:15 ;
过氧化氢酶:15;
亚硒酸钠:35.5;
1%二巯基乙醇溶液:1.5ml/L;
余量为水。
制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH 值至7.0~7.4,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
实施例3 制备人脂肪干细胞无血清培养基
各原料浓度如下,各浓度单位若无特别说明,均为mg/L:
L-精氨酸 305;
CuSO4·5H2O 0.005;
L-天门冬酰胺28;
L-天门冬氨酸12;
L-谷氨酸 35;
Ni(NO3)2·6H2O 0.0003;
L-谷氨酰胺0;
ZnSO4·7H2O 0.065;
甘氨酸 20;
CoCl2·6H2O 0.008;
L-组氨酸5;
NaSiO3·9H2O 0.001;
L-异亮氨酸 75;
Na3VO4·12H2O 0.005;
L-赖氨酸盐酸25;
SnCl2·2H2O 0.00001;
L-蛋氨酸35;
Na2SeO3 0.01;
L-苯丙氨酸5;
FeSO4·7H2O 0.1;
L-脯氨酸30;
葡萄糖 4000;
L-丝氨酸10;
维生素C 0.19;
L-苏氨酸30;
对羟基苯甲酸1.8;
L-色氨酸5;
丙酮酸钠 40;
L-缬氨酸30;
亚油酸 0.04;
L-亮氨酸 20;
β-巯基乙醇 0.5;
二水L-酪氨酸二钠420;
乙醇胺 5;
L-胱氨酸二盐酸20;
地塞米松 0.0015;
人转铁蛋白 45;
人血清白蛋白 5500;
纤粘连蛋白 0.5;
重组人成纤维细胞生长因子 0.1;
重组人转化因子β15;
重组人表皮生长因子15;
胰岛素:5;
纯化人转铁蛋白:1;
胆固醇: 55;
过氧化氢酶:45;
亚硒酸钠:15.3;
1%二巯基乙醇溶液:0.25ml/L;
余量为水。
制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH 值至7.0~7.4,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
实验
将实施例2制备的培养基置于T25细胞培养瓶中,将脂肪干细胞以1.0×104cells/cm2接种,在温度37℃下培养,统计原代细-胞的长出时间,每代的生长时间。
同时,以lonza无血清培养基(市场购买得到)为对照培养脐脂肪干细胞。
结论:
原代细胞收获时间:通常的细胞培养基进行酶消化法分离细胞,7~9天可收获原代细胞,而本发明的无血清培养基爬出细胞时间缩短至6~8天,提高了效率。
细胞每代生长时间,经过连续10代监测,平均每代生长时间在2~3天,比lonza无血清培养基培养时间缩短了一天,节约了培养时间,提高了工作效率。
连续传代次数:传统的MSC无血清培养基传代至6代以后,细胞生长变缓慢,本发明的培养基可连续传代至10代,细胞仍保持其分化潜能,而lonza培养基传代至第8代,细胞生长明显变缓,导致后期无法传代。
上述虽然结合实施例对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (5)
1.一种人脂肪干细胞无血清培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸 105~305;
CuSO4·5H2O 0.0006~0.005;
L-天门冬酰胺28~70;
L-天门冬氨酸12~32;
L-谷氨酸 15~35;
Ni(NO3)2·6H2O 0.000032~0.0003;
L-谷氨酰胺0~500;
ZnSO4·7H2O 0.065~0.65;
甘氨酸 5~20;
CoCl2·6H2O 0.001~0.008;
L-组氨酸5~35;
NaSiO3·9H2O 0.001~0.01;
L-异亮氨酸 20~75;
Na3VO4·12H2O 0.0005~0.005;
L-赖氨酸盐酸25~50;
SnCl2·2H2O 0.00001~0.0001;
L-蛋氨酸15~35;
Na2SeO3 0.002~0.01;
L-苯丙氨酸5~25;
FeSO4·7H2O 0.1~1.5;
L-脯氨酸5~30;
葡萄糖 1000~4000;
L-丝氨酸10~40;
维生素C 0.19~0.7;
L-苏氨酸10~30;
对羟基苯甲酸0.3~1.8;
L-色氨酸5~20;
丙酮酸钠 40~500;
L-缬氨酸15~30;
亚油酸 0.015~0.04;
L-亮氨酸 20~65;
β-巯基乙醇 0.5~3.5;
二水L-酪氨酸二钠120~420;
乙醇胺0.5~5;
L-胱氨酸二盐酸20~65;
地塞米松 0.0015~0.0045;
人转铁蛋白 5~45;
人血清白蛋白 1000~5500;
纤粘连蛋白 0.5~5;
重组人成纤维细胞生长因子 0.1~15;
重组人转化因子β 0.1~15;
重组人表皮生长因子0.1~15;
胰岛素:5~20;
纯化人转铁蛋白液:1~25;
胆固醇:15~55;
过氧化氢酶:15~45;
亚硒酸钠:15.3~35.5;
1%(质量百分数)二巯基乙醇溶液:0.25~1.5ml/L;
余量为水。
2.根据权利要求1所述的人脂肪干细胞无血清培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位为mg/L:
L-精氨酸 210;
CuSO4·5H2O 0.002;
L-天门冬酰胺55;
L-天门冬氨酸20;
L-谷氨酸 25;
Ni(NO3)2·6H2O 0.00012;
L-谷氨酰胺300;
ZnSO4·7H2O 0.32;
甘氨酸 12;
CoCl2·6H2O 0.0055;
L-组氨酸15;
NaSiO3·9H2O 0.006;
L-异亮氨酸 45;
Na3VO4·12H2O 0.0015;
L-赖氨酸盐酸35;
SnCl2·2H2O 0.000045;
L-蛋氨酸20;
Na2SeO3 0.006;
L-苯丙氨酸18;
FeSO4·7H2O 1.0;
L-脯氨酸20;
葡萄糖 3000;
L-丝氨酸25;
维生素C 0.450;
L-苏氨酸25;
对羟基苯甲酸1.0;
L-色氨酸15;
丙酮酸钠 250;
L-缬氨酸20;
亚油酸 0.025;
L-亮氨酸 45;
β-巯基乙醇 2.2;
二水L-酪氨酸二钠280;
乙醇胺 2.5;
L-胱氨酸二盐酸45;
地塞米松 0.002;
人转铁蛋白 30;
人血清白蛋白 2200;
纤粘连蛋白 2.5;
重组人成纤维细胞生长因子 5;
重组人转化因子β 5;
重组人表皮生长因子5;
胰岛素:10;
纯化人转铁蛋白:20;
胆固醇:30;
过氧化氢酶:25 ;
亚硒酸钠:20.5;
1%二巯基乙醇溶液:0.65ml/L;
余量为水。
3.根据权利要求1所述的人脂肪干细胞无血清培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位为mg/L:
L-精氨酸 105;
CuSO4·5H2O 0.0006;
L-天门冬酰胺70;
L-天门冬氨酸32;
L-谷氨酸 15;
Ni(NO3)2·6H2O 0.000032;
L-谷氨酰胺500;
ZnSO4·7H2O 0.65;
甘氨酸 5;
CoCl2·6H2O 0.001;
L-组氨酸35;
NaSiO3·9H2O 0.01;
L-异亮氨酸 20;
Na3VO4·12H2O 0.0005;
L-赖氨酸盐酸50;
SnCl2·2H2O 0.0001;
L-蛋氨酸15;
Na2SeO3 0.002;
L-苯丙氨酸25;
FeSO4·7H2O 1.5;
L-脯氨酸5;
葡萄糖 1000;
L-丝氨酸40;
维生素C 0.7;
L-苏氨酸10;
对羟基苯甲酸0.3;
L-色氨酸20;
丙酮酸钠 500;
L-缬氨酸15;
亚油酸 0.015;
L-亮氨酸 65;
β-巯基乙醇 3.5;
二水L-酪氨酸二钠120;
乙醇胺 0.5;
L-胱氨酸二盐酸65;
地塞米松 0.0045;
人转铁蛋白 5;
人血清白蛋白 1000;
纤粘连蛋白 5;
重组人成纤维细胞生长因子 15;
重组人转化因子β 0.1;
重组人表皮生长因子0.1;
胰岛素: 20;
纯化人转铁蛋白: 25;
胆固醇:15 ;
过氧化氢酶:15;
亚硒酸钠:35.5;
1%二巯基乙醇溶液:1.5ml/L;
余量为水。
4.权利要求1~3中任一项所述的人脂肪干细胞无血清培养基的制备方法,其特征在于:取除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如权利要求1~3中任一项所述,调节pH 值至7.0~7.4,即得。
5.权利要求1~3中任一项所述的人脂肪干细胞无血清培养基在培养人脂肪干细胞中的应用。
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CN112795532A (zh) * | 2019-11-13 | 2021-05-14 | 苏州易迈吉生物医药科技有限公司 | 一种人牙周膜干细胞无血清培养基 |
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CN112795532A (zh) * | 2019-11-13 | 2021-05-14 | 苏州易迈吉生物医药科技有限公司 | 一种人牙周膜干细胞无血清培养基 |
CN111518764A (zh) * | 2020-06-04 | 2020-08-11 | 广州同康生物科技有限公司 | 一种脂肪干细胞无血清培养基及其制备方法 |
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