CN111484970B - 一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基 - Google Patents
一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基 Download PDFInfo
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Abstract
本发明涉及细胞培养技术领域，特别涉及一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基。该无血清培养基由NaHCO3、亚硒酸钠、乙二胺四乙酸铁、L‑抗坏血酸、硫酸锌、重组人碱性成纤维细胞生长因子、重组人转化生长因子β、小分子抑制剂SB‑216763、小分子抑制剂5‑AzaC、叶酸和基础培养基组成。本发明提供的无血清培养基成分明确、无动物源、蛋白含量极低，能够实现ESCs快速增殖的同时维持细胞的未分化状态。
Description
技术领域
本发明涉及细胞培养技术领域，特别涉及一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基。
背景技术
胚胎干细胞(Embryonic stem cells，ESCs)主要从着床前的囊胚内细胞团(innercell mass，ICM)中分离获得，是一种多能干细胞，可以分化成体内几乎所有的细胞类型细胞、形成人体的组织和器官。1981年Kaufman和Evans首次建立了小鼠胚胎干细胞系，这一重大的科学进展使围绕胚胎干细胞的研究成为了科学家们的研究热点并取得更大的突破。1998年Tomson等首次成功分离得到了人类胚胎干细胞；2008年，科学家成功分离得到了大鼠胚胎干细胞。2016年中国科学院周琪院士的团队成功将小鼠胚胎干细胞体外分化成具有功能的精子细胞并产生可育后代。2018年澳大利亚的研究人员成功用干细胞修复帕金森患者脑中受损的多巴胺神经元。2019年1月9日世界首例人胚胎干细胞分化功能细胞治疗半月板损伤在华中科技大学同济医学院附属同济医院康复医学科顺利完成。
胚胎干细胞具有无限增殖、自我更新和多向分化的潜能，在体外特定的培养条件下能够不断的进行自我增殖并保持细胞的未分化状态，同时还可以分化为三胚层(内胚层、中胚层和外胚层)中的任何一种细胞类型。因此，ESCs在理论研究、临床应用等方面都具有广阔的应用前景，(1)可作为研究动物细胞生长分化和组织器官发育构建的有力工具；(2)用于建立体外疾病模型以便研究疾病的形成机制；(3)药物筛选和开发，体外将ESCs诱导分化为特定的细胞类型后加药物处理，研究药物对特定组织或细胞的毒性作用；(4)细胞治疗，如产生特定的细胞来替代各种疾病患者受损的组织或器官，从而用于治疗糖尿病、肝病、帕金森疾病和脊髓损伤等疾病；(5)基因治疗。
ESCs巨大的应用前景，决定了建立一种能够保证ESCs数量和质量的培养体系成为一种迫切的需求。胚胎干细胞的培养主要经历有血清有饲养层培养、无血清有饲养层培养和无血清无饲养层培养三个阶段。这些培养方法或多或少存在一些缺陷。有饲养层的培养体系中，ESCs的质量往往受到饲养层细胞状态的影响；饲养层细胞指能够分泌细胞外基质、黏附因子和各种生长因子，起到促进ESCs贴壁、增殖并抑制其分化作用的细胞。鼠胚胎成纤维细胞(MEF)、包皮成纤维细胞、间充质干细胞等均可作为ESCs培养的饲养层，其中MEF由于取材方便、抑分化效果好、易于分离等特点成为最为常用的饲养层。虽然饲养层细胞能够为ESCs提供较为理想的微环境，但是其携带的动物源蛋白和病原体易引起机体免疫反应。此外，饲养层细胞存在批次间的差异，制备过程繁琐，需要耗费大量的人力、物力和时间投入。血清同样存在成分不明确，含有大量的外源性物质和微生物感染(如病毒、支原体和内毒素等)等问题。这些因素影响研究实验的重复性，同时大大限制了ESCs在临床上的应用。
为了克服饲养层和血清带来的问题，很多研究者一直尝试建立一种无血清、无饲养层的ESCs培养体系。无血清、无饲养层培养体系指使用细胞外基质替代饲养层的基础上，采用无血清培养基对ESCs进行培养的体系。细胞外基质主要由细胞合成并分泌的大分子组成，如糖胺聚糖、蛋白多糖、胶原蛋白和黏连蛋白等，这些大分子影响着ESCs的贴壁、增殖和形态等。ESCs体外培养常用的胞外基质有基质胶、明胶、胶原、层黏连蛋白、纤连蛋白等。无血清培养基(SerμM-Free MediμM，SFM)指在基础培养基的基础上添加能够代替血清功能的各种因子形成的培养基，这些添加因子主要包括激素(如胰岛素、生长激素和胰高血糖素等)、生长因子、白蛋白、转铁蛋白、微量元素和维生素等。
目前，有不少的企业已经开发了各具特色的无血清、无饲养层ESCs培养基；目前市面上接受度最大的培养基基本上都是国外的品牌，如Stem Cell公司的mTeSRTM1、mTeSRTMPlus和TeSRTM-E8TM等。虽然市面上这些无血清培养基可以在一定程度上克服血清和饲养层细胞引发的问题，但是大多存在增殖能力不足、容易出现自分化和核型异常等问题；此外，这些培养基大多含有转铁蛋白、白蛋白和多种生长因子等生物大分子，高蛋白含量导致培养基成本居高不下，严重限制了科学研究和细胞制备企业的大规模使用。
发明内容
有鉴于此，本发明提供一种无血清、无饲养层的人胚胎干细胞培养基及其制备方法和胚胎干细胞培养方法。该培养基成分明确、无动物源、蛋白含量极低，能够实现ESCs快速增殖的同时维持细胞的未分化状态。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种无血清培养基，该无血清培养基由NaHCO3、亚硒酸钠、乙二胺四乙酸铁、L-抗坏血酸、硫酸锌、重组人碱性成纤维细胞生长因子、重组人转化生长因子β、小分子抑制剂SB-216763、小分子抑制剂5-AzaC、叶酸和基础培养基组成。
小分子抑制剂SB-216763是一种GSK3抑制剂，通过阻止Wnt信号通路中β-Catenin的降解导致细胞内β-Catenin积累，从而促进ESCs的自我更新，研究表明，经过SB-216763小分子处理的细胞，可以较好的上调胚胎干细胞多能性基因SSEA-1、Nanog的表达。小分子抑制剂5-AzaC是一种DNA甲基化转移酶抑制剂，可以使ESCs多能性标志基因的启动子区域去甲基化，上调多能性基因的表达，从而维持ESCs的干性。叶酸是一种水溶性维生素，是B族维生素家族中的一员，在人体多种重要的生理过程中都扮演着重要的角色。叶酸能够通过甲基化修饰促进Meg3基因的表达，增强LYAR蛋白的稳定性，进而增强ESCs的增殖能力和干性稳定性。乙二胺四乙酸铁可以作为转铁蛋白的替代物。硫酸锌则可以替代胰岛素的功能参与葡萄糖摄取、脂质代谢、蛋白质和核酸的合成。
本发明针对上述现有的无血清培养基存在的问题，将上述化合物应用于ESCs的无血清培养基中，减少培养基中蛋白质的含量，开发一种蛋白含量极低、成本更低、细胞增殖效果更好并且能够维持ESCs未分化状态的无血清无饲养层培养基。
作为优选，该无血清培养基中各组分用量如下：
优选地，该无血清培养基中各组分用量如下：
优选地，该无血清培养基中各组分用量如下：
更优选地，该无血清培养基中各组分用量如下：
作为优选，基础培养基为DMEM/F12培养基。
本发明还提供了该无血清培养基的制备方法，包括：将NaHCO3、亚硒酸钠、乙二胺四乙酸铁、L-抗坏血酸、硫酸锌、重组人碱性成纤维细胞生长因子、重组人转化生长因子β、小分子抑制剂SB-216763、小分子抑制剂5-AzaC、叶酸溶解于基础培养基，过滤除菌。
作为优选，过滤除菌所用滤膜的孔径为0.2～0.3μM。
在本发明提供的具体实施例中，过滤除菌所用滤膜的孔径为0.22μM。
本发明还提供了一种胚胎干细胞的培养方法，采用上述无血清培养基培养胚胎干细胞。
作为优选，胚胎干细胞为第7～9代胚胎干细胞，培养的密度为(1～10)×104cell/cm2。
在本发明提供的具体实施例中，胚胎干细胞为第8代胚胎干细胞，培养的密度为5×104cell/cm2。
本发明提供了一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基。该无血清培养基由NaHCO3、亚硒酸钠、乙二胺四乙酸铁、L-抗坏血酸、硫酸锌、重组人碱性成纤维细胞生长因子、重组人转化生长因子β、小分子抑制剂SB-216763、小分子抑制剂5-AzaC、叶酸和基础培养基组成。本发明具有的技术效果如下：
本发明提供的无血清培养基主要包含小分子抑制剂SB-216763、小分子抑制剂SB-216763、叶酸、乙二胺四乙酸铁和硫酸锌等10种原料，成分明确，无动物源，蛋白含量低，原料易得和配制简单方便。该培养基添加的小分子抑制剂SB-216763、小分子抑制SB-216763和叶酸对ESCs增殖、干性维持、克隆形成具有促进作用，解决了现有无血清培养基增殖能力不足和体外培养容易出现分化的问题。而乙二胺四乙酸铁和硫酸锌的添加减少了培养基的蛋白质含量，增加培养基性能稳定性的同时降低了培养基的成本。
附图说明
图1示ESCs形态图；
图2示ESCs扩增倍数；
图3示ESCs活率结果(％)；
图4示ESCs多能性基因mRNA表达水平。
具体实施方式
本发明公开了一种低蛋白含量的无血清无饲养层胚胎与多能干细胞培养基，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中，所涉及的组分、试剂均为常规市售产品。DMEM/F12购自Gibco公司。其他组分均可从sigma、MP、Gibco等公司购得。
下面结合实施例，进一步阐述本发明：
实施例1.无血清培养基配制
本实施例以DMEM/F12为基础培养基的无血清培养基成分包括：
上述成分按照各自的溶解特性溶解，0.22μM滤膜过滤除菌后加入DMEM/F12为基础培养基中混匀。
对比例1.无血清培养基配制
本对比例以DMEM/F12为基础培养基的无血清培养基成分包括：
上述成分按照各自的溶解特性溶解，0.22μM滤膜过滤除菌后加入DMEM/F12为基础培养基中混匀。
对比例2.无血清培养基配制
本对比例以DMEM/F12为基础培养基的无血清培养基成分包括：
上述成分按照各自的溶解特性溶解，0.22μM滤膜过滤除菌后加入DMEM/F12为基础培养基中混匀。
试验例
本发明将实施例1的含有小分子抑制剂SB-216763和5-AzaC的无血清培养基设为实验组1，将对比例1的仅含有小分子抑制剂SB-216763(无5-AzaC)的无血清培养基设为实验组2；将对比例2的仅含有小分子抑制剂5-AzaC(无SB-216763)的无血清培养基设为实验组3；将含10％FBS的DMEM/F12和Stem Cell公司的TeSRTM-E8TM培养基分别设为对照组1和对照组2，进行以下实验：
试验例1 ESCs形态观察
将第8代ESCs按照5×104cell/cm2的密度接种于纤连蛋白包被好的6孔板中，分别加入上述5组培养基进行培养，培养条件为37℃、5％CO2培养箱，第2天开始每天换液，培养至第5天，倒置显微镜下观察各组ESCs形态和采集图像。
如图1所示，对照组1、对照组2、实验组1细胞均呈典型的集落状态，克隆紧密，边缘界限清晰，但是与对照组1和对照组2相比，实验组1克隆面积更大，形态更规则，细胞间隙更致密。实验组2和实验组3克隆边缘出现不同程度的分化现象，且克隆形状不规则。说明本发明所述的无血清培养基具有维持ESCs正常形态和生长的效果，单一的小分子抑制剂可能无法有效维持ESCs典型的形态。
试验例2 ESCs增殖活性检测
将第8代ESCs按照5×104cell/cm2的密度接种于纤连蛋白包被好的6孔板中，分别加入上述5组培养基进行培养，第2天开始每天换液，培养至第5天，收集计数计算各组细胞扩增倍数及细胞活率，结果如图2及图3所示。
图2结果表明，与对照组1和对照组2相比，实验组1的扩增倍数明显升高，实验组2和实验组3细胞扩增效果最差。图3为ESCs的活性检测结果，该结果表明实验组1细胞活性高于其他任何组。说明本发明所述的无血清培养基具有较好的促进细胞增殖和维持细胞活率的效果。
试验例3 ESCs多能性基因检测
将第8代ESCs按照5×104cell/cm2的密度接种于纤连蛋白包被好的6孔板中，分别加入上述5组培养基进行培养，第2天开始每天换液，培养至第5天。用0.25％胰酶溶液消化后分别提取各组ESCs总RNA，并反转录为cDNA，以cDNA为模板，检测ESCs多能性基因Oct4、Sox2和Nanog的表达水平，引物序列见表1。
表1.ESCs多能性基因qPCR引物序列
如图4所示，与对照组1和对照组2相比，实验组1ESCs多能性基因mRNA的表达水平明显升高，虽然实验组2和实验组3虽然表达多能性基因，但表达量明显较低。表明本发明所述的无血清培养基能更好的维持ESCs的多能性和具有更强的自我更新能力。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一种无血清培养基，其特征在于，由NaHCO3、亚硒酸钠、乙二胺四乙酸铁、L-抗坏血酸、硫酸锌、重组人碱性成纤维细胞生长因子、重组人转化生长因子β、小分子抑制剂SB-216763、小分子抑制剂5-AzaC、叶酸和基础培养基组成；
各组分用量如下：
NaHCO3 400 mg/L
亚硒酸钠 30μg/L
乙二胺四乙酸铁 300μM
L-抗坏血酸 55mg/L
硫酸锌 30μg/L
重组人碱性成纤维细胞生长因子 50μg/L
重组人转化生长因子β 10μg/L
小分子抑制剂SB-216763 10μM
小分子抑制剂5-AzaC 10μM
叶酸 45 mg/L
基础培养基 补足。
2.根据权利要求1所述的无血清培养基，其特征在于，所述基础培养基为DMEM/F12培养基。
3.如权利要求1至2中任一项无血清培养基的制备方法，其特征在于，包括：将NaHCO3、亚硒酸钠、乙二胺四乙酸铁、L-抗坏血酸、硫酸锌、重组人碱性成纤维细胞生长因子、重组人转化生长因子β、小分子抑制剂SB-216763、小分子抑制剂5-AzaC、叶酸溶解于基础培养基，过滤除菌。
4.根据权利要求3所述的制备方法，其特征在于，所述过滤除菌所用滤膜的孔径为0.2~0.3μm。
5.一种非诊断治疗目的的胚胎干细胞的培养方法，其特征在于，采用权利要求1至2中任一项无血清培养基培养胚胎干细胞。
6.根据权利要求5所述的培养方法，其特征在于，所述胚胎干细胞为第7~9代胚胎干细胞，所述培养的密度为（1~10）×104cell/cm2。
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