CN114854679A - 脐带间充质干细胞扩增培养的方法 - Google Patents
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Abstract
本发明属于干细胞培养技术领域,具体涉及一种脐带间充质干细胞扩增培养的方法。针对现有脐带间充质干细胞分离培养方法效率低、培养得到的脐带间充质干细胞活性低,培养周期长等缺陷,本发明提供了一种脐带间充质干细胞扩增培养的方法,在分离脐带间充质干细胞时采用无血清培养基清洗,配合采用胶原酶和胰蛋白酶进行消化,分离效果好,得到的脐带间充质干细胞活性高;同时,本发明采用特制的贴壁培养基对细胞进行培养,能够使细胞贴壁生长更快更好,扩增培养效率高,培养得到的细胞活性高,应用范围更为广泛。本发明为脐带间充质干细胞扩增培养提供了一种又快又好的方法,适宜推广使用。
Description
技术领域
本发明属于干细胞培养技术领域,具体涉及一种脐带间充质干细胞扩增培养的方法。
背景技术
间充质干细胞(Mesenchymal stem cells,MSCs)是一种来源于发育早期中胚层的具有高度自我更新能力和多向分化潜能的多能干细胞,广泛存在于全身多种组织中,可在体外培养扩增,并能在特定条件下分化成神经细胞、成骨细胞、软骨细胞、肌肉细胞及脂肪细胞等。研究表明,在骨髓、肌肉、胃肠道、皮肤、视网膜、外周及中枢神经系统等组织中均存在间充质干细胞,间充质干细胞已经成为细胞替代治疗的研究热点。
脐带是间充质干细胞的分离来源之一,其中从人脐带中分离扩增培养脐带间充质干细胞是一种重要方式。目前,此类干细胞常用的分离培养方法是酶消化法和组织块贴壁培养法。酶消化法可以获得大量原代细胞,但是由于提取过程中消化酶的破坏,细胞贴壁效果、形态较差,并且组织块的消化时间在大规模生产过程中难于统一量化,另外分离提取成本较高,处理时间相对较长。而传统组织块培养法的缺点是原代培养时间普遍较长,需要15天左右。因此,目前脐带间充质干细胞的分离培养扩增方法还存在扩增效率低下,扩增得到的干细胞活性差,扩增培养周期长等诸多问题,急需开发一种能高效产生大量活性高的脐带间充质干细胞扩增培养方法。
发明内容
本发明要解决的技术问题为:现有脐带间充质干细胞分离培养方法效率低、培养得到的脐带间充质干细胞活性低,培养周期长等缺陷。
本发明解决上述技术问题的技术方案为:提供了一种脐带间充质干细胞扩增培养的方法。该方法包括以下步骤:
a、脐带间充质干细胞的分离
去除血管,将脐带组织切成小块,用无血清培养基清洗2~3次,浸泡于0.1~0.5%胶原酶中,37℃消化4~6h;
加入占消化液体积2~3倍的生理盐水,2000~2500r/min离心5~10min,弃上清,加入2.5~3.0%的胰蛋白酶,37℃处理30~40min;
再加入占消化液体积2~3倍的生理盐水,100目滤网过滤,2000~2500r/min离心5~10min,弃上清,分离得到脐带间充质干细胞;
b、脐带间充质干细胞扩增培养
采用步骤a的无血清培养基,将步骤a得到的细胞调成悬液,按2×104个/cm2接种于培养瓶中,置于37℃,5%CO2条件下培养4~5天,去除非贴壁细胞;
向贴壁细胞中加入贴壁培养基,每2~3天换液,观察细胞融合至80~90%时,采用消化液进行消化,按1:3传代,培养15~20天;
采用胰酶消化细胞,细胞悬液2000~2500r/min离心5~10min,弃上清液,加入生理盐水混匀,2000~2500r/min离心5~10min,重复2次,获得扩增后的脐带间充质干细胞。
其中,上述脐带间充质干细胞扩增培养的方法中,步骤a所述的脐带组织切成1~3cm的小块。
其中,上述脐带间充质干细胞扩增培养的方法中,步骤a所述的无血清培养基为不含动物源性成分和人血小板裂解物的培养基。
其中,上述脐带间充质干细胞扩增培养的方法中,步骤b所述的消化液为0.25%胰蛋白酶和1mmol/L EDTA的混合物。
其中,上述脐带间充质干细胞扩增培养的方法中,步骤b所述的贴壁培养基的组成包括:基础培养基,表皮细胞生长因子,氨基酸,维生素,葡萄糖,低氧诱导因子-1,胰岛素,生姜提取物,神经酰胺,青霉素和链霉素。
进一步的,上述脐带间充质干细胞扩增培养的方法中,所述的基础培养基为DMEM、α-MEM、F12、DMEM/F12、RPMI1640或IMEM中的至少一种。
进一步的,上述脐带间充质干细胞扩增培养的方法中,所述的贴壁培养基的组成为:基础培养基,表皮细胞生长因子20~30ng/mL,氨基酸20~30μg/mL,维生素15-30μg/mL,葡萄糖40-60μg/mL,低氧诱导因子-110~20ng/mL,胰岛素15~20ug/mL,生姜提取物1~2ng/mL,神经酰胺10~15μg/mL,青霉素30~50U/mL和链霉素20~30U/mL。
其中,上述脐带间充质干细胞扩增培养的方法中,步骤b所述的充质干细胞高表达CD73、CD90和CD105,同时低表达或不表达CD34、CD45、HLA-DR、CD11b和CD19。
本发明的有益效果为:
本发明提供了一种脐带间充质干细胞扩增培养的方法,在分离脐带间充质干细胞时采用无血清培养基清洗,配合采用胶原酶和胰蛋白酶进行消化,分离效果好,得到的脐带间充质干细胞活性高;同时,本发明采用特制的贴壁培养基对细胞进行培养,能够使细胞贴壁生长更快更好,扩增培养效率高,培养得到的细胞活性高,应用范围更为广泛。本发明为脐带间充质干细胞扩增培养提供了一种又快又好的方法,适宜推广使用。
具体实施方式
本发明提供了一种脐带间充质干细胞扩增培养的方法,包括以下步骤:
a、脐带间充质干细胞的分离
去除血管,将脐带组织切成小块,用无血清培养基清洗2~3次,浸泡于0.1~0.5%胶原酶中,37℃消化4~6h;
加入占消化液体积2~3倍的生理盐水,2000~2500r/min离心5~10min,弃上清,加入2.5~3.0%的胰蛋白酶,37℃处理30~40min;
再加入占消化液体积2~3倍的生理盐水,100目滤网过滤,2000~2500r/min离心5~10min,弃上清,分离得到脐带间充质干细胞;
b、脐带间充质干细胞扩增培养
采用步骤a的无血清培养基,将步骤a得到的细胞调成悬液,按2×104个/cm2接种于培养瓶中,置于37℃,5%CO2条件下培养4~5天,去除非贴壁细胞;
向贴壁细胞中加入贴壁培养基,每2~3天换液,观察细胞融合至80~90%时,采用消化液进行消化,按1:3传代,培养15~20天;
采用胰酶消化细胞,细胞悬液2000~2500r/min离心5~10min,弃上清液,加入生理盐水混匀,2000~2500r/min离心5~10min,重复2次,获得扩增后的脐带间充质干细胞。
在本发明的扩增培养中,最为关键的是采用了特制的贴壁培养基,其组成包括:基础培养基,表皮细胞生长因子,氨基酸,维生素,葡萄糖,低氧诱导因子-1,胰岛素,生姜提取物,神经酰胺,青霉素和链霉素。
本发明使用的培养基为无血清培养基,培养基各成分完全不含动物来源组分,克服了异源蛋白和污染源的缺点,且所有成分明确,符合临床级别干细胞药物的要求。在所述培养基中,基础培养基用于提供细胞基础代谢所需的无机盐、氨基酸和葡萄糖。同时,本发明还特别添加了氨基酸和葡萄糖,增加基础培养基养分。同时,本发明还添加了胰岛素,可提高脐带间充质干细胞的合成代谢能力,刺激细胞的生长增殖;还添加了维生素,其是细胞的生长代谢的必需物质,能够促进细胞生长、延长细胞活性,减少代谢产物堆积;并且,本发明还加入了低氧诱导因子-1,其对脐带间充质干细胞增殖起到了促进作用,改善了其贴壁性能,贴壁效果更好。此外,本发明还加入了表皮细胞生长因子,促进间充质干细胞的生长增殖。更为特别的是,本发明还加入了生姜提取物,提高细胞活性,增加细胞活跃度。神经酰胺,促进间充质干细胞代谢,减少细胞老化,延长细胞寿命。
本发明通过上述精心设计的配方,能够激活间充质干细胞的活力潜能,提高细胞的活性和分泌能力,并且能够选择性的促进间充质干细胞体外培养中的生长和分化,并为其达到最理想营养平衡状态提供数量上和质量上的保证,显著提高了细胞增殖速度。
下面将通过实施例对本发明的具体实施方式做进一步的解释说明,但不表示将本发明的保护范围限制在实施例所述范围内。
实施例和对比例中所使用的设备和试剂均为普通市售产品,人脐带组织来源于四川大学生物治疗国家重点实验室,已获国际伦理学组织批准。
实施例1采用本发明方法扩增培养脐带间充质干细胞
具体的操作步骤如下:
a、脐带间充质干细胞的分离
去除血管,将脐带组织切成小块,用无血清培养基清洗2次,浸泡于0.1%胶原酶中,37℃消化4h;
加入占消化液体积2倍的生理盐水,2000r/min离心5min,弃上清,加入2.5%的胰蛋白酶,37℃处理30min;
再加入占消化液体积2倍的生理盐水,100目滤网过滤,2000r/min离心5min,弃上清,分离得到脐带间充质干细胞;
b、脐带间充质干细胞扩增培养
采用步骤a的无血清培养基,将步骤a得到的细胞调成悬液,按2×104个/cm2接种于培养瓶中,置于37℃,5%CO2条件下培养4天,去除非贴壁细胞;
向贴壁细胞中加入贴壁培养基,每2天换液,观察细胞融合至80~90%时,采用消化液进行消化,按1:3传代,培养15天;所述的贴壁培养基组成为:基础培养基,表皮细胞生长因子20ng/mL,氨基酸20μg/mL,维生素15μg/mL,葡萄糖40μg/mL,低氧诱导因子-110ng/mL,胰岛素15ug/mL,生姜提取物1ng/mL,神经酰胺10μg/mL,青霉素30U/mL和链霉素20U/mL;
用胰酶消化细胞,细胞悬液2000r/min离心5min,弃上清液,加入生理盐水混匀,2000r/min离心5min,重复2次,获得扩增后的脐带间充质干细胞。
实施例2采用本发明方法扩增培养脐带间充质干细胞
具体的操作步骤如下:
a、脐带间充质干细胞的分离
去除血管,将脐带组织切成小块,用无血清培养基清洗3次,浸泡于0.5%胶原酶中,37℃消化6h;
加入占消化液体积3倍的生理盐水,2500r/min离心10min,弃上清,加入3.0%的胰蛋白酶,37℃处理40min;
再加入占消化液体积3倍的生理盐水,100目滤网过滤,2500r/min离心10min,弃上清,分离得到脐带间充质干细胞;
b、脐带间充质干细胞扩增培养
采用步骤a的无血清培养基,将步骤a得到的细胞调成悬液,按2×104个/cm2接种于培养瓶中,置于37℃,5%CO2条件下培养5天,去除非贴壁细胞;
向贴壁细胞中加入贴壁培养基,每3天换液,观察细胞融合至80~90%时,采用消化液进行消化,按1:3传代,培养20天;所述的贴壁培养基组成为:基础培养基,表皮细胞生长因子30ng/mL,氨基酸30μg/mL,维生素30μg/mL,葡萄糖60μg/mL,低氧诱导因子-120ng/mL,胰岛素20ug/mL,生姜提取物2ng/mL,神经酰胺15μg/mL,青霉素50U/mL和链霉素30U/mL;
用胰酶消化细胞,细胞悬液2500r/min离心10min,弃上清液,加入生理盐水混匀,2500r/min离心10min,重复2次,获得扩增后的脐带间充质干细胞。
实施例3采用本发明方法扩增培养脐带间充质干细胞
具体的操作步骤如下:
a、脐带间充质干细胞的分离
去除血管,将脐带组织切成小块,用无血清培养基清洗2次,浸泡于0.3%胶原酶中,37℃消化5h;
加入占消化液体积3倍的生理盐水,2200r/min离心8min,弃上清,加入3.0%的胰蛋白酶,37℃处理40min;
再加入占消化液体积2倍的生理盐水,100目滤网过滤,2200r/min离心8min,弃上清,分离得到脐带间充质干细胞;
b、脐带间充质干细胞扩增培养
采用步骤a的无血清培养基,将步骤a得到的细胞调成悬液,按2×104个/cm2接种于培养瓶中,置于37℃,5%CO2条件下培养5天,去除非贴壁细胞;
向贴壁细胞中加入贴壁培养基,每2天换液,观察细胞融合至80~90%时,采用消化液进行消化,按1:3传代,培养20天;所述的贴壁培养基组成为:基础培养基,表皮细胞生长因子25ng/mL,氨基酸25μg/mL,维生素30μg/mL,葡萄糖50μg/mL,低氧诱导因子-115ng/mL,胰岛素15ug/mL,生姜提取物1.5ng/mL,神经酰胺12μg/mL,青霉素40U/mL和链霉素25U/mL;
用胰酶消化细胞,细胞悬液2200r/min离心8min,弃上清液,加入生理盐水混匀,2200r/min离心8min,重复2次,获得扩增后的脐带间充质干细胞。
对比例1不采用本发明方法扩增培养脐带间充质干细胞
与实施例3的区别在于:贴壁培养基的组成仅为基础培养基。
对比例2不采用本发明方法扩增培养脐带间充质干细胞
与实施例3的区别在于:贴壁培养基的组成为:基础培养基,表皮细胞生长因子25ng/mL,低氧诱导因子-115ng/mL,胰岛素15ug/mL,生姜提取物1.5ng/mL,神经酰胺12μg/mL,青霉素40U/mL和链霉素25U/mL。
对比例3不采用本发明方法扩增培养脐带间充质干细胞
与实施例3的区别在于:贴壁培养基的组成为:基础培养基,表皮细胞生长因子25ng/mL,氨基酸25μg/mL,维生素30μg/mL,葡萄糖50μg/mL,低氧诱导因子-115ng/mL,青霉素40U/mL和链霉素25U/mL。
对比例4不采用本发明方法扩增培养脐带间充质干细胞
与实施例3的区别在于:贴壁培养基的组成为:基础培养基,表皮细胞生长因子25ng/mL,氨基酸25μg/mL,维生素30μg/mL,葡萄糖50μg/mL,低氧诱导因子-115ng/mL,胰岛素15ug/mL,生姜提取物1.5ng/mL,神经酰胺12μg/mL。对比例5不采用本发明方法扩增培养脐带间充质干细胞
与实施例3的区别在于:贴壁培养基的组成为:基础培养基,表皮细胞生长因子50ng/mL,氨基酸10μg/mL,维生素50μg/mL,葡萄糖100μg/mL,低氧诱导因子-15ng/mL,胰岛素10ug/mL,生姜提取物0.5ng/mL,神经酰胺20μg/mL,青霉素20U/mL和链霉素10U/mL。
对实施例和对比例扩增培养的细胞采用流式细胞仪检测,计算细胞的增殖指数,结果如表1所示,增殖指数的计算公式如下:PI=(S+G2/M)/(G0/G1+S+G2/M)×100%(其中G0、G1、S、G2、M为细胞的生长的五个周期)。
表1不同方法扩增的脐带间充质干细胞增殖指数
增殖指数(%) | |
实施例1 | 53.5 |
实施例2 | 55.1 |
实施例3 | 57.2 |
对比例1 | 32.9 |
对比例2 | 42.7 |
对比例3 | 45.3 |
对比例4 | 44.0 |
对比例5 | 48.9 |
由表1的结果可看出,采用本发明的贴壁培养基,能够提高间充质干细胞的贴壁生长能力,细胞增殖指数显著增加。
分别将实施例1~3和对比例1~5的培养基加入T75培养瓶中,接种复苏后的脐带充质干细胞,细胞浓度为2×104个/mL,置于37℃,5%CO2的培养箱中培养,每2天传代一次,传代比例为1:3,培养7d后采用台盼蓝染色法统计脐带间充质干细胞的存活率,结果如表2所示。
表2不同培养基对脐带间充质干细胞的存活率的影响
细胞存活率(%) | |
实施例1 | 96.2 |
实施例2 | 95.4 |
实施例3 | 98.3 |
对比例1 | 77.5 |
对比例2 | 84.0 |
对比例3 | 82.7 |
对比例4 | 87.4 |
对比例5 | 89.9 |
由上述实验结果可知,本发明的贴壁培养基由于添加了多种成分,各种成分间协同增效,有助于细胞保持活性,减少细胞凋亡,进而提高细胞的存活率。
综上可见,本发明提供了一种脐带间充质干细胞的扩增培养方法,采用特制的贴壁培养基,能够快速、高效的获得大量的脐带间充质干细胞,得到的脐带间充质干细胞活性高,存活率高,能够满足细胞治疗的需求。
Claims (8)
1.脐带间充质干细胞扩增培养的方法,其特征在于,包括以下步骤:
a、脐带间充质干细胞的分离
去除血管,将脐带组织切成小块,用无血清培养基清洗2~3次,浸泡于0.1~0.5%胶原酶中,37℃消化4~6h;
加入占消化液体积2~3倍的生理盐水,2000~2500r/min离心5~10min,弃上清,加入2.5~3.0%的胰蛋白酶,37℃处理30~40min;
再加入占消化液体积2~3倍的生理盐水,100目滤网过滤,2000~2500r/min离心5~10min,弃上清,分离得到脐带间充质干细胞;
b、脐带间充质干细胞扩增培养
采用步骤a的无血清培养基,将步骤a得到的细胞调成悬液,按2×104个/cm2接种于培养瓶中,置于37℃,5%CO2条件下培养4~5天,去除非贴壁细胞;
向贴壁细胞中加入贴壁培养基,每2~3天换液,观察细胞融合至80~90%时,采用消化液进行消化,按1:3传代,培养15~20天;
采用胰酶消化细胞,细胞悬液2000~2500r/min离心5~10min,弃上清液,加入生理盐水混匀,2000~2500r/min离心5~10min,重复2次,获得扩增后的脐带间充质干细胞。
2.根据权利要求1所述的脐带间充质干细胞扩增培养的方法,其特征在于:步骤a所述的脐带组织切成1~3cm的小块。
3.根据权利要求1所述的脐带间充质干细胞扩增培养的方法,其特征在于:步骤a所述的无血清培养基为不含动物源性成分和人血小板裂解物的培养基。
4.根据权利要求1所述的脐带间充质干细胞扩增培养的方法,其特征在于:步骤b所述的消化液为0.25%胰蛋白酶和1mmol/L EDTA的混合物。
5.根据权利要求1所述的脐带间充质干细胞扩增培养的方法,其特征在于:步骤b所述的贴壁培养基的组成包括:基础培养基,表皮细胞生长因子,氨基酸,维生素,葡萄糖,低氧诱导因子-1,胰岛素,生姜提取物,神经酰胺,青霉素和链霉素。
6.根据权利要求5所述的脐带间充质干细胞扩增培养的方法,其特征在于:所述的基础培养基为DMEM、α-MEM、F12、DMEM/F12、RPMI1640或IMEM中的至少一种。
7.根据权利要求5所述的脐带间充质干细胞扩增培养的方法,其特征在于:所述的贴壁培养基的组成为:基础培养基,表皮细胞生长因子20~30ng/mL,氨基酸20~30μg/mL,维生素15-30μg/mL,葡萄糖40-60μg/mL,低氧诱导因子-110~20ng/mL,胰岛素15~20ug/mL,生姜提取物1~2ng/mL,神经酰胺10~15μg/mL,青霉素30~50U/mL和链霉素20~30U/mL。
8.根据权利要求1所述的脐带间充质干细胞扩增培养的方法,其特征在于:步骤b所述的充质干细胞高表达CD73、CD90和CD105,同时低表达或不表达CD34、CD45、HLA-DR、CD11b和CD19。
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