CN111996162B - 一种成软骨分化培养基及其应用 - Google Patents

一种成软骨分化培养基及其应用 Download PDF

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CN111996162B
CN111996162B CN202010935091.3A CN202010935091A CN111996162B CN 111996162 B CN111996162 B CN 111996162B CN 202010935091 A CN202010935091 A CN 202010935091A CN 111996162 B CN111996162 B CN 111996162B
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stem cells
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于宝利
李其雷
陈旭
陈刚
杨建国
孙芳
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Suzhou Ecosai Biotechnology Co ltd
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Abstract

本发明涉及一种成软骨分化培养基及其应用，由基础培养基和添加组分组成，成分包括无机盐组分、氨基酸组分、维生素组分、微量元素组分和碳水化合物以及TGF‑beta3、BMP‑6、BMP‑2、人血白蛋白、地塞米松、L‑抗坏血酸‑2‑磷酸酯镁盐、重组人胰岛素、重组人转铁蛋白、硒酸、亚油酸。本发明还提供了间充质干细胞成软骨无血清分化培养基的用途。本发明成分明确，不含有动物源成分，不添加血清，更安全，且在各组分协同作用可以高效的诱导间充质干细胞向软骨细胞的分化。

Description

一种成软骨分化培养基及其应用
技术领域
本发明属于属于细胞培养技术领域，具体为一种成软骨分化培养基。
背景技术
干细胞是一类具有高度自我更新和多向分化潜能的细胞。根据所在发育阶段，可分为胚胎干细胞和成体干细胞。干细胞研究目前是生物医药行业热点研究方向之一，由于胚胎干细胞涉及伦理道德问题，现成体干细胞的研究和应用已成为主流。间充质干细胞作为一类成体干细胞具有来源丰富、高度自我增殖多向分化、低免疫源性等优势，已成为细胞治疗的理想种子细胞。
由于细胞治疗的特殊性，用于细胞治疗的间充质干细胞的鉴定越来越受到重视，国家有6号)和《干细胞制剂质量控制及临床前研究指导原则(试行)》明确提出可通过检测干细胞分化潜能、诱导分化细胞的结构和生理功能、对免疫细胞的调节能力、分泌特定细胞因子、表达特定基因和或蛋白等功能，判断干细胞制剂与治疗相关的生物学有效性。对间充质干细胞，无论何种来源，应进行体外多种类型细胞(如成脂肪细胞、成软骨细胞、成骨细胞等)分化能力的检测，以判断其细胞分化的多能性。
目前也有效果较好的商业化培养基，如STEMCELL公司的成软骨分化培养基产品(STEMCELL，05455)，分化效果优良，但是该培养基成本高昂。
以血清为主要营养成分的传统成软骨分化培养基，诱导分化速度慢、效率低，成分不明确，分化效果受血清批次及质量影响，更由于血清的存在带来潜在人畜共患病风险，对于操作者及分化细胞后细胞的进一步科学研究和应用研究带来了安全风险。
发明内容
本发明的第一个目的是，针对现有技术的不足，针对现有软骨分化培养基分化效率低、分化时间长，生物安全性低的不足，本发明提供更安全、高效的干细胞成软骨分化无血清培养基。
本发明的第二个目的是，提供干细胞成软骨分化无血清培养基的用途。
为了实现上述第一个目的，本发明采用的技术方案是：
基础培养基和添加组分组成，基础培养基由无机盐组分、氨基酸组分、维生素组分、微量元素组分和碳水化合物组成，添加组分由重组人转化生长因子-β3(TGF-beta3)、重组人骨形态发生蛋白6(BMP-6)、重组人骨形态发生蛋白2(BMP-2)、降钙素(Calcitonin)、人血白蛋白、地塞米松、L-抗坏血酸-2-磷酸酯镁盐、重组人胰岛素、重组人转铁蛋白、硒酸、亚油酸组成。
优选的，添加组分以体积计，各成分的含量为：重组人转化生长因子-β3：25.0ug/L、重组人骨形态发生蛋白6：0.25mg/L、重组人骨形态发生蛋白2：0.25mg/L、降钙素1.25mg/L、人血白蛋白112.5g/L、地塞米松25uM/L、L-抗坏血酸-2-磷酸酯镁盐1.25g/L、重组人胰岛素1.875g/L、重组人转铁蛋白2.5g/L、硒酸3.75ug/L、亚油酸125mg/L；
基础培养基与添加组分的体积比为100：1～100：10。
优选的，基础培养基与添加组分的体积比为100：4。
优选的，基础培养基，成分组成如下：
为了实现上述第二个目的，本发明采用的技术方案是：
成软骨分化培养基用于脂肪、骨髓、脐带、骨骼肌等来源的间充质干细胞体外诱导骨髓间充质干细胞分化的用途。
作为本发明的一种优先实施方案：间充质干细胞成成软骨分化培养基用于间充质干细胞体外诱导成软骨分化的用途。
由于上述技术方案的运用，本发明与现有技术相比具有以下优点：
本发明通过独特的基础培养基配方设计，更适用于间充质干细胞的培养；
本发明的培养基内添加重组人血白蛋白及羧甲基纤维素钠等成分，不含有人源提取物质成分，不含有动物源成分，成分明确，不需要进行艾滋病病毒(HIV-1/2)抗体、乙肝表面抗原(HBsAg)抗体和丙肝病毒(HCV)检测，同时可排除人畜共患病风险，使用更安全、便利，减少质控需求，下游细胞诱导分化应用更安全。
本发明的培养基添加多种脂肪酸组合，配合多种细胞因子3-异丁基-1-甲基黄嘌呤、重组人重组人胰岛素生长因子、地塞米松、匹格列酮盐酸盐协同作用，能够快速的在体外诱导间充质干细胞向成熟脂肪细胞分化，分化效率更高，可以更短时间内检测到分化细胞的形成。
本发明所用的基础培养基根据细胞分化培养过程中的成分消耗情况进行成分优化设计，在细胞生长必需物质基础上，增加了成软骨分化所需营养物质的添加量，更适用于细胞成软骨分化的需求。
本发明的成软骨分化无血清培养基在基础培养基基础上，添加TGF-beta3、BMP-6、BMP-2、Calcitonin、人血白蛋白、地塞米松、L-抗坏血酸-2-磷酸酯镁盐、重组人胰岛素、重组人转铁蛋白、硒酸、亚油酸成分，各成分相互作用，可以高效的诱导间充质干细胞向软骨细胞的分化，特别是TGF-beta3、BMP-6、BMP-2、Calcitonin的组合应用，TGF-beta3促进细胞的分化过程，BMP-6、BMP-2促进细胞的成软骨分化，Calcitonin进一步提升BMP-6、BMP-2因子的促分化作用，可以实现间充质干细胞高效的分化为软骨细胞，在14-21天即可达到很好的分化效果。
本发明的间充质干细胞成软骨分化无血清培养基，成分明确，不含有血清，不含有动物源成分，可排除人畜共患病风险，使用更安全、便利，减少质控需求，下游细胞诱导分化应用更安全。
附图说明
图1是本发明实施例2中，本发明所述间充质干细胞成软骨分化无血清培养基诱导骨髓间充质干细胞成软骨分化的检测结果，及商业化成软骨分化培养基(含血清)分化结果。
图2是本发明实施例3中，培养基添加不同配比的基础培养基与添加组分，诱导骨髓间充质干细胞成软骨分化的检测结果。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明
作进一步说明。
正如背景介绍中介绍的，成软骨分化能力是间充质干细胞的特点之一，而如何快速、高效、安全的诱导间充质干细胞成软骨分化成为间充质干细胞研究应用的重要一环。为了提高间充质干细胞成软骨分化的速度、效率和安全性，需要研发出一种间充质干细胞成软骨分化无血清培养基。本发明通过优化设计基础培养基，并添加多种细胞生长因子、多种蛋白质等成分，替代血清的同时，达到快速、高效、安全的诱导间充质干细胞向软骨细胞分化。
实施例1
本实施例提供的间充质干细胞成软骨分化无血清培养基由以下成分组成：基础培养基和添加组分。
(1)基础培养基标记为M1，按如下方案配制：
Figure BDA0002671649250000041
Figure BDA0002671649250000051
Figure BDA0002671649250000061
上述各种组分均可从Sigma、阿拉丁等试剂公司购得，各组分溶于1L注射水内，以0.22微米滤膜过滤，即得到基础培养基M1。
(2)添加组分记为S2组分，S2组分物质组成如下：
Figure BDA0002671649250000062
本例中，地塞米松、亚油酸可溶于无水乙醇，Calcitonin、重组人胰岛素可溶于0.05M的盐酸溶液，其他组分均可溶于注射水，以上各成分溶解后，按上表添加量混合，以0.22微米滤膜过滤，即得到S2溶液。
本例中，间充质干细胞软骨分化培养基以M1与S2按体积比1000mL:40mL配制而成。
对比例1
为商品化成脂分化培养基(STEMCELL，05455)。
实施例2
本实施例提供实施例1的间充质干细胞成软骨分化无血清培养基的效果验证。本实施例的实验中采用人骨髓间充质干细胞，该细胞来源于ATCC标准细胞库(PCS-500-012，Lot 70011720，ATCC)，此细胞用于下述所有实验。
一、实验方法
1.人骨髓间充质干细胞的培养
间充质干细胞无血清培养基(Excell Bio，ME000-N023)，复苏人骨髓间充质干细胞(ATCC，PCS-500-012，Lot 70011720，Passage 5)，以8000/cm2密度接种于100mm细胞培养皿。
添加间充质干细胞无血清培养基(Excell Bio，ME000-N023)，进行培养，添加量约为15ml，每48h更换一次培养基，骨髓间充质干细胞生长至约85％丰度(约72～96h)时收获细胞。
2.分化培养
1)试验组设置：实施例1、对比例1培养基分别设置实验组，每组设置3个重复组；
2)经传代收集的间充质干细胞，计数，300g离心5分钟，使用所述间充质干细胞成软骨分化无血清培养基重悬细胞，并使细胞密度达到1.6×107/mL；
3)将5ul细胞悬液滴在12孔板中央；
4)对照组使用含血清成软骨分化培养基重悬细胞，以相同的密度接种细胞；
5)37℃，5％CO2，饱和湿度环境下静置2小时；
6)每孔加入1mL对应培养基，每3天更换新鲜培养基，培养14天；
7)4％甲醛溶液固定。
3.染色鉴定
1)在通风厨内弃去固定液，并用DPBS润洗一次；
2)每孔加入0.5mL用0.1N HCl配制的1％阿利辛蓝溶液(Sigma，Catalog#66011-100mL-F)，室温染色30min；
3)弃去染液，并用0.1N HCl润洗三次，；
4)加入蒸馏水中和酸液，显微镜下观察、分析。
检测结果参见图1所示，结果显示，采用本实施例1的间充质干细胞成软骨分化培养基，细胞经过14天的诱导培养，可成功诱导骨髓间充质干细胞分化为软骨细胞。
实施例3
本实施例提供不同配比效果测试，按照本例实施例1的方案配制基础培养基(M1)和添加组分(S2)，基础培养基与添加组分按照不同配比进行间充质干细胞成脂分化培养基的配制，并检验各配比培养基的成软骨诱导分化的效果。诱导分化14天后进行阿辛蓝染色。
本实施例使用的间充质干细胞及培养方法与实施例2中相同。
1)培养基组
Figure BDA0002671649250000081
Figure BDA0002671649250000091
按照上表配比配制M1～M5培养基，
2)细胞培养
按照实施例2相同的方案培养骨髓间充质干细胞，经传代收集的间充质干细胞，计数，300g离心5分钟，使用所述间充质干细胞成软骨分化无血清培养基重悬细胞，并使细胞密度达到1.6×107/mL，将5ul细胞悬液滴在12孔板中央，细胞贴壁后启动诱导分化。
3)成软骨诱导分化及鉴定
本例设置5个试验组：M1～M5；
向培养板内分别加入M1～M5培养基1mL，每孔加入1mL培养基，每组设置3个重复组，每3天更换新鲜培养基，培养14天，进行染色鉴定。
结果显示，如附图2所示，M2、M3、M4均可诱导骨髓间充质干细胞向软骨细胞分化，其中M3分化效果最好，可以形成较大软骨球，M2、M4部分细胞也形成软骨球，并被染色；M1培养分化效果较差，无软骨球形成，仅有少量细胞分化被染色；M5培养基细胞大量致死，无分化细胞。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，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 (3)

1.一种成软骨分化培养基，其特征在于，由基础培养基和添加组分组成，基础培养基由无机盐组分、氨基酸组分、维生素组分、微量元素组分和碳水化合物组成，添加组分由重组人转化生长因子-β3、重组人骨形态发生蛋白6、重组人骨形态发生蛋白2、降钙素、人血白蛋白、地塞米松、L-抗坏血酸-2-磷酸酯镁盐、重组人胰岛素、重组人转铁蛋白、硒酸、亚油酸组成；
所述的添加组分以体积计，各成分的含量为：重组人转化生长因子-β3：25.0ug/L、重组人骨形态发生蛋白6：0.25mg/L、重组人骨形态发生蛋白2：0.25mg/L、降钙素1.25mg/L、人血白蛋白112.5g/L、地塞米松25uM/L、L-抗坏血酸-2-磷酸酯镁盐1.25g/L、重组人胰岛素1.875g/L、重组人转铁蛋白2.5g/L、硒酸3.75ug/L、亚油酸125mg/L；
所述基础培养基与添加组分的体积比为100∶1～100∶10；
所述的基础培养基，成分组成如下：
Figure FDA0003287519280000011
Figure FDA0003287519280000021
Figure 1
Figure 2
2.根据权利要求1所述的成软骨分化培养基在间充质干细胞或胚胎干细胞的成软骨分化中的用途。
3.根据权利要求2所述的成软骨分化培养基在间充质干细胞或胚胎干细胞的成软骨分化中的用途，其特征在于，所述的间充质干细胞来源于脂肪、骨髓、牙髓、脐带、胎盘中的任一组织。
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