CN109652364A - 一种人羊膜上皮细胞的无血清培养基 - Google Patents
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Abstract
本发明公开了一种人羊膜上皮细胞的无血清培养基，包括上皮细胞基础培养基和添加剂，以上皮细胞基础培养基的体积作为基准，所述添加剂组成及其含量分别为纤连蛋白，体积比2％‑5％，转铁蛋白，体积比为5％‑10％，胰岛素2‑10mg/L，L‑谷氨酰胺，2‑4mM，腐胺，1‑10mg/L，黄体酮10‑15ng/mL，葡萄糖，5‑25mM，脂类复合物，2‑4mM，微量元素，1‑5mg/L。本发明采用无血清等动物源的成分，以DMEM/F12基础培养基以及添加剂两大部分组成，能够有效地避免因使用血清带来的诸多的不利影响，无血清培养基内部添加人表皮生长因子以及适当添加促贴壁物质、促生长因子以及酶抑制剂等添加物，延长人羊膜上皮细胞在体外的寿命，有效地扩增其数量，此外，保持其干细胞和上皮细胞的特性，提高干细胞和上皮细胞的质量。
Description
技术领域
发明涉及生物技术领域，具体为一种人羊膜上皮细胞的无血清培养基。
背景技术
干细胞移植凭借其细胞来源丰富、不易感染、免疫原性低和并发症少等优点，已逐渐成为当今生物医学研究的热点。人羊膜上皮细胞是位于人胎盘羊膜上立方体柱状紧密排列的单层细胞，表达多种胚胎干细胞表面标记，是一类具有自我更新与多向分化潜能的成体干细胞，具有不表达端粒酶、无致瘤性、免疫原性低以及安全性好等优点，因此具有广阔的临床应用前景。
但是，目前对于人羊膜上皮细胞体外培养过程中其增殖以及分裂速度较胚胎干细胞要慢，因此在实际的人羊膜上皮细胞体外培养过程中其增殖分化的能力较差，导致扩增数量不足，此外，传统的对于人羊膜上皮细胞体外培养使用的培养基是含有牛血清的培养基，由于血清成分复杂易带入外源病毒等问题易对人羊膜上皮细胞体外培养增加阻碍，此外，市面上存在的无血清培养基无法很好地适应人羊膜上皮细胞体外扩增时的特点，进而无法明确培养物的成分以及促进人羊膜上皮细胞的顺利分化，为此，我们提出了一种人羊膜上皮细胞的无血清培养基。
发明内容
发明的目的在于提供一种人羊膜上皮细胞的无血清培养基，以解决上述背景技术中提出的问题。
为实现上述目的，发明提供如下技术方案：一种人羊膜上皮细胞的无血清培养基，其特征在于：以上皮细胞基础培养基的体积作为基准，所述添加剂组成及其含量分别为纤连蛋白，体积比2％-5％，转铁蛋白，体积比为5％-10％，胰岛素2-10mg/L，L-谷氨酰胺，2-4mM，腐胺，1-10mg/L，黄体酮10-15ng/mL，葡萄糖，5-25mM，脂类复合物，2-4mM，微量元素，1-5mg/L。
优选的，所述上皮细胞基础培养基为DMEM/F12(1：1，V/V)基础培养基。
优选的，所述无血清培养基的PH值控制在6.5-7.5之间。
优选的，所述无血清培养基另添加有人表皮生长因子(EGF)1-10ng/mL。
优选的，所述无血清培养基另外添加2-4mg/L的大豆胰酶作为酶抑制剂。
优选的，所述上皮细胞基础培养基和添加剂按照特定体积比或含量混合在人羊膜上皮细胞培养之前构成人羊膜上皮细胞无血清培养体系。
优选的，所述人表皮生长因子(EGF)为人羊膜上皮细胞培养过程中加入人羊膜上皮细胞无血清培养体系内。
与现有技术相比，发明的有益效果是：
1、该种人羊膜上皮细胞的无血清培养基，采用无血清等动物源的成分，以DMEM/F12基础培养基以及添加剂两大部分组成，基础培养基含有各种营养物质能够很好的供人羊膜上皮细胞体外扩增过程中一系列的生命活动，同时，能够有效地避免因使用血清带来的诸多的不利影响。
2、该种人羊膜上皮细胞的无血清培养基，无血清培养基内部添加人表皮生长因子以及适当添加促贴壁物质、促生长因子以及酶抑制剂等添加物，改变培养基整体成分及形态，同时，延长人羊膜上皮细胞在体外的寿命，保护人羊膜上皮细胞在体外扩增过程，有效地扩增其数量，此外，保持其干细胞和上皮细胞的特性，保证了干细胞的质量。
具体实施方式
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，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例一：
发明提供一种技术方案：一种人羊膜上皮细胞的无血清培养基，其特征在于：以上皮细胞基础培养基的体积作为基准，所述添加剂组成及其含量分别为纤连蛋白，体积比2％，转铁蛋白，体积比为5％，胰岛素2mg/L，L-谷氨酰胺，2mM，腐胺，1mg/L，黄体酮10ng/mL，葡萄糖，5mM，脂类复合物，2mM，微量元素，1mg/L。
所述上皮细胞基础培养基为DMEM/F12(1：1，V/V)基础培养基。
所述无血清培养基的PH值控制在6.5。
所述无血清培养基另添加有人表皮生长因子(EGF)1ng/mL。
所述无血清培养基另外添加2mg/L的大豆胰酶作为酶抑制剂。
所述上皮细胞基础培养基和添加剂按照特定体积比或含量混合在人羊膜上皮细胞培养之前构成人羊膜上皮细胞无血清培养体系。
所述人表皮生长因子(EGF)为人羊膜上皮细胞培养过程中加入人羊膜上皮细胞无血清培养体系内。
实施例二：
发明提供一种技术方案：一种人羊膜上皮细胞的无血清培养基，其特征在于：以上皮细胞基础培养基的体积作为基准，所述添加剂组成及其含量分别为纤连蛋白，体积比5％，转铁蛋白，体积比为10％，胰岛素10mg/L，L-谷氨酰胺，4mM，腐胺，10mg/L，黄体酮15ng/mL，葡萄糖，25mM，脂类复合物，4mM，微量元素，5mg/L。
优选的，所述上皮细胞基础培养基为DMEM/F12(1：1，V/V)基础培养基。
优选的，所述无血清培养基的PH值控制在7.5之间。
优选的，所述无血清培养基另添加有人表皮生长因子(EGF)10ng/mL。
优选的，所述无血清培养基另外添加4mg/L的大豆胰酶作为酶抑制剂。
优选的，所述上皮细胞基础培养基和添加剂按照特定体积比或含量混合在人羊膜上皮细胞培养之前构成人羊膜上皮细胞无血清培养体系。
优选的，所述人表皮生长因子(EGF)为人羊膜上皮细胞培养过程中加入人羊膜上皮细胞无血清培养体系内。
实施例三：
发明提供一种技术方案：一种人羊膜上皮细胞的无血清培养基，其特征在于：以上皮细胞基础培养基的体积作为基准，所述添加剂组成及其含量分别为纤连蛋白，体积比3.5％，转铁蛋白，体积比为7.5％，胰岛素5mg/L，L-谷氨酰胺，3mM，腐胺，5mg/L，黄体酮12.5ng/mL，葡萄糖，15mM，脂类复合物，3mM，微量元素，3mg/L。
优选的，所述上皮细胞基础培养基为DMEM/F12(1：1，V/V)基础培养基。
优选的，所述无血清培养基的PH值控制在7之间。
优选的，所述无血清培养基另添加有人表皮生长因子(EGF)5.5ng/mL。
优选的，所述无血清培养基另外添加3mg/L的大豆胰酶作为酶抑制剂。
优选的，所述上皮细胞基础培养基和添加剂按照特定体积比或含量混合在人羊膜上皮细胞培养之前构成人羊膜上皮细胞无血清培养体系。
优选的，所述人表皮生长因子(EGF)为人羊膜上皮细胞培养过程中加入人羊膜上皮细胞无血清培养体系内。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (7)
1.一种人羊膜上皮细胞的无血清培养基，包括上皮细胞基础培养基和添加剂，其特征在于：以上皮细胞基础培养基的体积作为基准，所述添加剂组成及其含量分别为纤连蛋白，体积比2％-5％，转铁蛋白，体积比为5％-10％，胰岛素2-10mg/L，L-谷氨酰胺，2-4mM，腐胺，1-10mg/L，黄体酮10-15ng/mL，葡萄糖，5-25mM，脂类复合物，2-4mM，微量元素，1-5mg/L。
2.如权利要求1所述的一种人羊膜上皮细胞的无血清培养基，其特征在于：所述上皮细胞基础培养基为DMEM/F12(1：1，V/V)基础培养基。
3.如权利要求1所述的一种人羊膜上皮细胞的无血清培养基，其特征在于：所述无血清培养基的PH值控制在6.5-7.5之间。
4.如权利要求1所述的一种人羊膜上皮细胞的无血清培养基，其特征在于：所述无血清培养基另添加有人表皮生长因子(EGF)1-10ng/mL。
5.如权利要求1所述的一种人羊膜上皮细胞的无血清培养基，其特征在于：所述无血清培养基另外添加2-4mg/L的大豆胰酶作为酶抑制剂。
6.如权利要求1所述的一种人羊膜上皮细胞的无血清培养基，其特征在于：所述上皮细胞基础培养基和添加剂按照特定体积比或含量混合在人羊膜上皮细胞培养之前构成人羊膜上皮细胞无血清培养体系。
7.如权利要求6或所述的一种人羊膜上皮细胞的无血清培养基，其特征在于：所述人表皮生长因子(EGF)为人羊膜上皮细胞培养过程中加入人羊膜上皮细胞无血清培养体系内。
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