CN112143695A - 一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基 - Google Patents
一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基 Download PDFInfo
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Abstract
本发明公开了一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基，该培养基由如下成分组成：基础培养基成分（表2）；血清添加物：10%FBS、0.4ul/mlEGF、4.5g/L D‑Glucose、1%L‑Glutamine、0.5ul/mlInsulin,human recombinant,zinc solution、10ul/mlMEM NEAA、110mg/L sodium Pyruvate。有益效果在于：本发明所述的培养基通过改变现有培养基的配比成分，不仅使得培养基的成分更全，价格低廉，能够适用于hMSC、hUSC、hDPSC、hADSC的单独培养，提高了培养基的功能性，而且能够在稳定干细胞的干性的同时，增加扩增速度、稳定细胞形态，有效缩短细胞培养周期，提高细胞培养效率。
Description
技术领域
本发明涉及到培养基领域，尤其涉及一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基。
背景技术
培养基，是指供给微生物、植物或动物(或组织)生长繁殖的，由不同营养物质组合配制而成的营养基质。一般都含有碳水化合物、含氮物质、无机盐(包括微量元素)、维生素和水等几大类物质。培养基既是提供细胞营养和促使细胞增殖的基础物质，也是细胞生长和繁殖的生存环境。培养基种类很多，根据配制原料的来源可分为自然培养基、合成培养基、半合成培养基；根据物理状态可分为固体培养基、液体培养基、半固体培养基；根据培养功能可分为基础培养基、选择培养基、加富培养基、鉴别培养基等；根据使用范围可分为细菌培养基、放线菌培养基、酵母菌培养基、真菌培养基等。
然而现有市面上的培养基组分不够全面，价格较高，一种培养基一般只能实现一种细胞的培养，使得培养基的功能性较差，且培养基的培养速度较慢，导致细胞的培养周期较长，降低了细胞培养效率。
发明内容
本发明的目的就在于为了解决上述问题而提供一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基。
本发明通过以下技术方案来实现上述目的：
一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基，该培养基由如下成分组成：该培养基由如下成分组成：基础培养基成分(表1)；血清添加物：10％FBS、0.4ul/mlEGF、4.5g/L D-Glucose、1％L-Glutamine、0.5ul/mlInsulin,human recombinant,zincsolution、10ul/mlMEM NEAA、110mg/L sodium Pyruvate。
进一步的，所述基础培养基与所述添加剂按照10:1的比例进行配制，能够使得培养基中成分更全，营养配比更加的合理。
所述的培养基的配制方法，包括如下步骤(以hUSCs为例)：
步骤(1)：将基础培养基与所述添加剂按照10:1的比例放置在容器中，并通过轻轻搅拌，以使得基础培养基与所述添加剂充分混合，制得培养基基液备用；
步骤(2)：向所述步骤(1)所制得的培养基积液中加入添加剂成分，并在加入的过程中保持不断搅拌，以确保两者充分溶解，待各成分混合均匀后，即可制得该培养基；
步骤(3)：原代培养时，该培养基的使用方法如下：
取材：接取≥100ml健康人中段尿于无菌尿瓶中，放于4℃冰箱短期保存，或立即处理。加入1％penicllin-Streptomycin。
离心：将尿瓶表面用酒精消毒后放入超净台，分装于无菌50ml离心管中，在4℃400rcf离心10min。
洗涤：离心后，弃去上清，用含有抗生素的45ml PBS洗涤，在4℃300rcf离心5min。重复洗涤两次。
种植：弃上清，分别用1ml完全培养基重悬细胞，按照培养瓶/皿要求加入培养基，放入37℃CO2恒温箱培养。
细胞扩增：培养3-5天后，可见多个单个细胞贴壁生长。每隔2天换液，培养至多个单细胞克隆达到70％-80％融合时，用0.25％胰蛋白酶消化约3min，血清终止消化，800rpm，5min离心，2ml培养基重悬细胞，转移瓶/皿中继续培养、传代和扩增。
传代后，按照1×10*5个/ml种植在175cm3大瓶，加入22ml上述培养基，显微镜下可见8小时细胞贴壁，12小时细胞开始扩增，48小时细胞密度为80％-90％，按上述方法继续传代，可传15代；
步骤(4)：hUSCs鉴定:
hPDSC、hUSC、hADSC、hMSC细胞贴壁12h贴壁，48h长满，且状态良好。
流式细胞术鉴定：
表达间质标志CD29、CD44、CD73、CD90、CD105，不表达成熟造血细胞标志CD45及内皮标志CD31和CD133，不表达HLA-DR。(以hUSCs为例)
表1不同代数hUSCs流式结果
分化鉴定：P2和P10成骨细胞、成软骨细胞、成脂肪细胞分化良好。
基础培养基成分表2如下：
相对于现有技术，本发明所述的培养基具有以下优势：
本发明所述的培养基通过改变现有培养基的配比成分，不仅使得培养基的成分更全，价格低廉，能够适用于hMSC、hUSC、hDPSC、hADSC的单独培养，提高了培养基的功能性，而且能够在稳定干细胞的干性的同时，增加扩增速度、稳定细胞形态，有效缩短细胞培养周期，提高细胞培养效率。
附图说明
图1为本发明所述的一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基中MSC在培养过程中的流式图；
图2为本发明所述的一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基中USC在培养过程中的流式图；
图3为本发明所述的一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基中DPSC在培养过程中的流式图；
图4为本发明所述的一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基中ADSC的流式图；
图5为本发明所述的一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基中hMSC、hUSC、hDPSC、hADSC时间与生长状态图；
图6为本发明所述的一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基中hMSC、hUSC、hDPSC、hADSC P2代增殖曲线图。
具体实施方式
一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基，该培养基由如下成分组成：基础培养基成分(表1)；血清添加物：10％FBS、0.4ul/mlEGF、4.5g/L D-Glucose 1％L-Glutamine、0.5ul/mlInsulin,human recombinant,zinc solution、10ul/mlMEM NEAA、110mg/L sodium Pyruvate。
优选的，所述的FBS能够为细胞培养通过营养环境；所述的EGF是一种细胞分离因子，能够促进细胞的分裂；所述的Glutamine为蛋白质合成中的编码氨基酸，能够促进细胞的分离、增殖；所述的Insulin,human recombinant,zinc solution可促进钾离子和镁离子穿过细胞膜进入细胞内，进而促进脱氧核糖核酸(DNA)、核糖核酸(RNA)及三磷酸腺苷(ATP)的合成，所述的NEAA中含有细胞培养所需的非必需氨基酸，能有效改善细胞培养基配比，降低细胞培养时细胞自身生产非必须氨基酸的副作用。
优选的，所述基础培养基和添加物按照10:1的比例进行配制，能够使得培养基中成分更全，营养配比更加的合理。
所述的培养基的配制方法，包括如下步骤：
步骤(1)：将基础培养基和添加物按照10:1的比例放置在容器中，并通过轻轻搅拌，以使得基础培养基和添加物充分混合，制得培养基基液备用；
步骤(2)：向所述步骤(1)所制得的培养基积液中加入添加物，并在加入的过程中保持不断搅拌，以确保添加物和培养基充分溶解，待各成分混合均匀后，即可制得该培养基。
优选的，该培养基的使用方法如下：使用时首先用1ml培养基重悬细胞，并按照培养瓶/皿要求将重悬后的细胞加入到培养基中，放入37℃CO2恒温箱培养；原代培养3-5天后(以尿来源干细胞为例)，可见多个单个细胞贴壁生长，每隔2天换液，培养至多个单细胞克隆达到70％-80％融合时，用0.25％胰蛋白酶消化约3min，血清终止消化，800rpm，5min离心，2ml完全培养基重悬细胞，转移瓶/皿中继续培养、传代和扩增。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (4)
1.一种可以用于hMSC、hUSC、hDPSC、hADSC的廉价高效培养基，其特征在于：该培养基由如下成分组成：基础培养基成分（表2）；血清添加物：10%FBS、0.4ul/mlEGF、4.5g/L D-Glucose 1% L-Glutamine、0.5ul/mlInsulin, human recombinant, zinc solution、10ul/mlMEM NEAA、110mg/L sodium Pyruvate。
2.根据权利要求1所述的培养基，其特征在于：所述基础培养基成分按照特定的比例进行配制。
3.权利要求1-2中任一项所述的培养基的配制方法，其特征在于：包括如下步骤：
步骤（1）：将基础培养基和添加剂按照10:1的比例放置在容器中，并通过轻轻搅拌，以使得基础培养基和添加物充分混合，制得培养基基液备用；
步骤（2）：向所述步骤（1）所制得的培养基积液中依次加入10%FBS、0.4ul/mlEGF、4.5g/L D-Glucose 1% L-Glutamine、0.5ul/mlInsulin, human recombinant, zinc solution、10ul/mlMEM NEAA、110mg/L sodium Pyruvate，并在加入的过程中保持不断搅拌，以确保10%FBS、0.4ul/mlEGF、4.5g/L D-Glucose 1% L-Glutamine、0.5ul/mlInsulin, humanrecombinant, zinc solution、10ul/mlMEM NEAA、110mg/L sodium Pyruvate充分溶解形成添加物；
将培养基成分和添加剂按照一定比例待各成分混合均匀后，即可制得该培养基。
4.根据权利要求3所述的培养基的配制方法，其特征在于：该培养基的使用方法如下：使用时首先用1 ml培养基重悬细胞，并按照培养瓶/皿要求将重悬后的细胞加入到培养基中，放入37℃ CO2恒温箱培养；原代培养3-5天后（以尿来源干细胞为例），可见多个单个细胞贴壁生长，每隔2天换液，培养至多个单细胞克隆达到70%-80%融合时，用0.25%胰蛋白酶消化约3 min，血清终止消化，800 rpm，5 min离心，2 ml培养基重悬细胞，转移瓶/皿中继续培养、传代和扩增。
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