CN112391340A - 一种间充质干细胞培养基 - Google Patents
一种间充质干细胞培养基 Download PDFInfo
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Abstract
本发明公开了一种间充质干细胞培养基，包括基础培养基和添加剂，基础培养基为α‑MEM培养基，添加剂为转铁蛋白、纤连蛋白、EGF、TGF‑β1、PDGF‑BB、胰岛素、胰蛋白酶、抑肽酶、氢化可的松、黄体酮、地塞米松、亚油酸、亚硒酸钠、卵磷脂、维A酸、维生素C、维生素E、维生素B7和维生素B12。通过应用以上技术方案，间充质干细胞培养基成分确定，质量可靠，成本低，并且体外培养扩增速度和细胞成活率高。
Description
技术领域
本发明属于生物技术领域，具体涉及一种间充质干细胞培养基。
背景技术
间充质干细胞(mesenchymal stem cells，MSCs)是中胚层来源的具有高度自我更新能力和多向分化潜能的多能干细胞，已从肌肉、脂肪、牙周质、脐血、脐带等多种组织中分离得到。经研究证实，MSCs免疫原性较低，不易引起机体的排斥反应；具有分化成其他中胚层细胞的潜能，能够分泌多种细胞因子；具有定向迁移能力。MSCs具有的这些优良特性使之在受损组织修复、器官移植、自体免疫性疾病治疗、肿瘤治疗等方面展现出良好的应用前景。
MSCs体外培养所用的培养基主要有血清培养基和无血清培养基，有血清培养基通常通过添加血清为细胞生长增殖提供所需的激素、生长因子等各种营养物质，但其不可控因素过多，无法保证其成分是否被病毒和支原体感染，血清中不确定的成分及配比更是会导致干细胞的干性丢失并走向分化，为细胞体外培养带来风险；无血清培养基一般由基础培养基和替代血清的添加剂两部分组成，能有效避免血清带来的诸多问题，也能满足体外细胞培养的要求。
传统的用于扩增MSCs的含血清培养基已不能满足临床需求，尽管现有技术中已经开发了几种适合MSCs体外扩增的无血清培养基，但是在使用时仍存在一定问题，如培养后细胞表面特异性抗原表达、分化潜能等有所下降、扩增速度和细胞成活率较低。究其原因，主要是对培养基中添加成分对MSCs的生物学行为的影响及相关机制认识有限。
因此，如何提供一种成分确定且质量可控的MSCs培养基，以提高MSCs体外培养扩增速度和细胞成活率是目前本领域技术人员亟待解决的技术问题。
发明内容
鉴于现有技术中MSCs体外扩增的无血清培养基存在的问题，本发明提供了一种间充质干细胞培养基，包括基础培养基和添加剂，所述基础培养基为α-MEM(minimum Eagle’smedium)培养基，所述添加剂组分为转铁蛋白、纤连蛋白、EGF(重组人表皮生长因子)、TGF-β1(重组人转化生长因子)、PDGF-BB(重组人血小板衍生生长因子)、胰岛素、胰蛋白酶、抑肽酶、氢化可的松、黄体酮、地塞米松、亚油酸、亚硒酸钠、卵磷脂、维A酸、维生素C、维生素E、维生素B7和维生素B12。
优选的，所述添加剂的具体含量为：转铁蛋白10-25μg/mL；纤连蛋白60-80μg/mL；EGF35-40ng/mL；TGF-β1 5-10ng/mL；PDGF-BB 50-60ng/mL；胰岛素1-10U/mL；胰蛋白酶10-20μg/mL；抑肽酶50-80μg/mL；氢化可的松1-1.5μg/mL；黄体酮10-20ng/mL；地塞米松0.5-1μmol/mL；亚油酸130-150μg/mL；亚硒酸钠20-30nmol/mL；卵磷脂50-60nmol/mL；维A酸0.2-0.5mmol/mL；维生素C 55μg/mL；维生素E 60μg/mL；维生素B7 15μg/mL；维生素B12 20μg/mL。
本发明公开了一种间充质干细胞培养基，与现有技术相比具有以下有益效果：
本发明间充质干细胞培养基无动物血清成分，成分确定，质量可靠，成本低；
本发明间充质干细胞培养基为干细胞培养提供了更为稳定的环境，进一步提升了体外培养扩增速度和细胞成活率。
附图说明
为了更清楚地说明本申请的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例中脐带间充质干细胞传代培养第3代第3天的生长状况。
具体实施方式
下面将结合本申请实施例中的附图，对本申请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申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
在下述实施例中，除特殊说明以外，所用试剂均为生化级试剂，并能通过商业渠道获得。下述实施例中所提及的细胞培养箱的培养条件，如无特殊说明，均为温度37℃，湿度95％，CO2体积分数5％。
实施例1
本实施例提供的一种间充质干细胞培养基，通过在α-MEM培养基中添加下述添加剂制备，具体添加剂组分和含量为：转铁蛋白20μg/mL；纤连蛋白80μg/mL；EGF 35ng/mL；TGF-β1 10ng/mL；PDGF-BB 60ng/mL；胰岛素5U/mL；胰蛋白酶15μg/mL；抑肽酶70μg/mL；氢化可的松1μg/mL；黄体酮15ng/mL；地塞米松0.5μmol/mL；亚油酸130μg/mL；亚硒酸钠25nmol/mL；卵磷脂50nmol/mL；维A酸0.2mmol/mL；维生素C55μg/mL；维生素E 60μg/mL；维生素B7 15μg/mL；维生素B12 20μg/mL。
实施例2
本实施例提供的一种间充质干细胞培养基，通过在α-MEM培养基中添加下述添加剂制备，具体添加剂组分和含量为：转铁蛋白25μg/mL；纤连蛋白70μg/mL；EGF 40ng/mL；TGF-β1 10ng/mL；PDGF-BB 55ng/mL；胰岛素5U/mL；胰蛋白酶15μg/mL；抑肽酶70μg/mL；氢化可的松1μg/mL；黄体酮20ng/mL；地塞米松0.5μmol/mL；亚油酸140μg/mL；亚硒酸钠30nmol/mL；卵磷脂50nmol/mL；维A酸0.3mmol/mL；维生素C55μg/mL；维生素E 60μg/mL；维生素B7 15μg/mL；维生素B12 20μg/mL。
实施例3
本实施例提供的一种间充质干细胞培养基，通过在α-MEM培养基中添加下述添加剂制备，具体添加剂组分和含量为：转铁蛋白25μg/mL；纤连蛋白60μg/mL；EGF 40ng/mL；TGF-β1 5ng/mL；PDGF-BB 60ng/mL；胰岛素5U/mL；胰蛋白酶15μg/mL；抑肽酶70μg/mL；氢化可的松1.5μg/mL；黄体酮15ng/mL；地塞米松0.5μmol/mL；亚油酸150μg/mL；亚硒酸钠25nmol/mL；卵磷脂55nmol/mL；维A酸0.5mmol/mL；维生素C55μg/mL；维生素E 60μg/mL；维生素B7 15μg/mL；维生素B12 20μg/mL。
下面将应用上述实施例中配制的培养基培养HUC-MSCs(人脐带间充质干细胞)。
(1)HUC-MSCs复苏培养
在50mL离心管中预先加入30mL复温至室温的α-MEM基础培养基，从液氮罐中取出一支冻存HUC-MSCs种子细胞的冻存管，在37℃水浴中快速融化后，将HUC-MSCs转移到离心管中，混合均匀后分别取出10mL悬液加入到3个新的50mL离心管中，离心后将上清弃掉；分别在3个离心管中加入15mL实施例1培养基、实施例2培养基和实施例3培养基，计数后调整细胞密度，接种至细胞培养皿或培养瓶中，在细胞培养箱静置培养。
(2)HUC-MSCs传代培养
当HUC-MSCs细胞达到90％汇合时，吸出培养基，用磷酸缓冲盐溶液(phosphatebuffer saline，PBS)清洗，加入适量0.25％胰蛋白酶消化2-5min，吸弃胰蛋白酶后加入每组相应的培养基将细胞吹散，离心后将上清弃掉，并补加每组相应的培养基，计数后调整细胞密度，接种至细胞培养皿或培养瓶中，在细胞培养箱继续静置培养。经镜下观察，各实施例中的细胞均达到90％以上融合率，细胞形态良好。图1是实施例1培养基培养的脐带间充质干细胞传代培养至第3代第3天的生长状况。
(3)流式细胞仪检测
收集实施例1-3的P7代HUC-MSCs，分别进行CytomicsTMFC500流式细胞仪检测。表1为检测结果，如表1结果所示，采用本发明培养基传代培养得到的P7代HUC-MSCs的CD105、CD73、CD90和HLA-ABC的表达量高于90％，CD45、CD34和HLA-DR低于1％，说明采用本发明培养基培养得到的P7代HUC-MSCs继续保持间充质干细胞的特性。
表1
对实施例1-3的P7代HUC-MSCs进行细胞计数，计数结果如表2所示，说明本发明培养基可以促进细胞增殖，而且细胞纯度更高。
表2
| 实施例1 | 实施例2 | 实施例3 | |
| 细胞数(个) | 2.89×10<sup>7</sup> | 2.93×10<sup>7</sup> | 2.96×10<sup>7</sup> |
以上公开的仅为本发明实施例的具体实施场景，并非是对本发明作其它形式的限制，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范作用，本申请实施例并非局限于此，任何熟悉本领域的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。上述序号仅仅为了描述，不代表实施场景的优劣。如非另行定义，文中所用的所有专业和科学用语与本领域技术人员所熟悉的意义相同。凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
Claims (1)
1.一种间充质干细胞培养基，其特征在于，包括基础培养基和添加剂，所述基础培养基为α-MEM培养基，所述添加剂的组分和含量为：
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| CN106190964A (zh) * | 2016-07-13 | 2016-12-07 | 中国科学院广州生物医药与健康研究院 | 一种间充质干细胞无血清培养基 |
| CN106906182A (zh) * | 2017-04-28 | 2017-06-30 | 北京赛斯达生物技术有限公司 | 一种间充质干细胞无血清培养基 |
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| CN113234671A (zh) * | 2021-05-08 | 2021-08-10 | 中山大学 | 全反式维甲酸的新应用 |
| CN113208998A (zh) * | 2021-05-11 | 2021-08-06 | 奥启(深圳)创投科技有限公司 | 一种含干细胞外泌体的抗皱精华液 |
| CN117802039A (zh) * | 2024-03-01 | 2024-04-02 | 泉美智能科技(山东)有限公司 | 一种无血清间充质干细胞三维培养基及其应用 |
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