CN110373384A - 一种无血清脂肪干细胞培养基及脂肪干细胞的培养方法 - Google Patents
一种无血清脂肪干细胞培养基及脂肪干细胞的培养方法 Download PDFInfo
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Abstract
本发明公开了一种无血清脂肪干细胞培养基及脂肪干细胞的培养方法,该培养基包括基础培养基和添加剂;所述基础培养基为DMEM/F12培养基,所述添加剂包括:转铁蛋白,成纤维细胞生长因子,C‑藻蓝素,刺五加皂苷B,矢车菊素‑3‑O‑葡萄糖苷,β‑蜕皮甾酮。本发明的培养基和培养方法既能促进脂肪干细胞的生长和增殖能力,又能降低培养过程中脂肪干细胞的增殖活性损伤,从而大幅度提高脂肪干细胞的体外扩增效率。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种无血清脂肪干细胞培养基及脂肪干细胞的培养方法。
背景技术
脂肪干细胞(adipose-derived stem cells,ADSCs)是从脂肪组织提取的一种成体干细胞,具有向软骨、骨、脂肪等多种组织分化的潜能。脂肪组织通过脂肪抽吸术或外科手术容易获取,对供者创伤较小且不涉及伦理问题。而且,脂肪干细胞在人和动物体内相对分布广泛、扩增操作简单方便,是医学和组织工程领域最具潜力的种子细胞之一。
目前,脂肪干细胞的体外培养主要采用含胎牛血清的培养基,由于含动物来源血清成分,存在引起排斥反应的潜在安全隐患,而且含血清培养基培养不同批次细胞的批间差异较大,增加了生产质量控制的难度。无血清培养基既能满足细胞在体外长时间培养的要求,又能避免含血清培养基带来的不利因素,成为近年来脂肪干细胞培养的研究热点。但是,无血清培养基中所含营养成分、促生长成分不足,而且无血清培养基缺乏血清中中和毒素、保护细胞的天然成分,导致脂肪干细胞的增殖活性受到损伤。由于这些因素的综合影响,使无血清培养基中的脂肪干细胞增殖速度较慢,很难获得足够临床使用的细胞数量,无法满足临床应用的需求。
发明内容
基于背景技术存在的技术问题,本发明提出了一种无血清脂肪干细胞培养基及脂肪干细胞的培养方法。
本发明提出的无血清脂肪干细胞培养基及脂肪干细胞的培养方法能促进脂肪干细胞的增殖,从而提高脂肪干细胞的体外扩增速度。
一种无血清脂肪干细胞培养基,包括基础培养基和添加剂;所述基础培养基为DMEM/F12培养基,所述添加剂包括:转铁蛋白,成纤维细胞生长因子,C-藻蓝素,刺五加皂苷B,矢车菊素-3-O-葡萄糖苷,β-蜕皮甾酮。
优选地,所述添加剂的终浓度为:转铁蛋白8-12μg/mL,成纤维细胞生长因子30-40ng/mL,C-藻蓝素2-3μg/mL,刺五加皂苷B 0.9-1.6μg/mL,矢车菊素-3-O-葡萄糖苷0.8-2.2μg/mL,β-蜕皮甾酮10-20μg/mL。
更优选地,所述添加剂的终浓度为:转铁蛋白10μg/mL,成纤维细胞生长因子38ng/mL,C-藻蓝素2.8μg/mL,刺五加皂苷B1.2μg/mL,矢车菊素-3-O-葡萄糖苷1.6μg/mL,β-蜕皮甾酮14μg/mL。
一种脂肪干细胞的培养方法,采用上述无血清脂肪干细胞培养基培养。
该脂肪干细胞的培养方法具体包括:将脂肪干细胞接种于培养皿中,加入所述无血清脂肪干细胞培养基进行培养,待细胞长到80-90%融合度后消化,按1:3-1:4比例传代。
优选地,该脂肪干细胞的培养方法具体包括:
S1、取脂肪组织,用PBS清洗后剪碎,加入0.1-0.15%的I型胶原酶消化15-20min,1200-1500r/min离心10-15min,然后用pH为7.2-7.4的D-Hank's液重悬,经过100-200目筛网过滤,1200-1500r/min离心10-15min;
S2、将步骤S1获得的细胞接种于培养皿中,加入所述无血清脂肪干细胞培养基进行培养,待细胞长到80-90%融合度后消化,按1:3-1:4比例传代。
优选地,所述脂肪干细胞的接种密度为1-1.5×104/cm2。
优选地,所述培养条件为37℃、CO2浓度5%。
优选地,所述步骤S2中,消化采用浓度为0.25%的胰酶-EDTA溶液。
优选地,所述培养皿在接种前,先用促进细胞贴壁的包被液进行包被,更优选地,所述包被液为质量浓度0.2-0.3%的明胶溶液。
本发明的有益效果如下:
本发明以DMEM/F12培养基为基础,加入转铁蛋白,成纤维细胞生长因子,C-藻蓝素,刺五加皂苷B,矢车菊素-3-O-葡萄糖苷,β-蜕皮甾酮作为添加剂,配制得到无血清脂肪干细胞培养基。其中,DMEM/F12培养基和转铁蛋白配合,为干细胞提供足够的营养,促进干细胞的生长;成纤维细胞生长因子、刺五加皂苷B和β-蜕皮甾酮配合,能促进干细胞的分裂增殖;C-藻蓝素,刺五加皂苷B和矢车菊素-3-O-葡萄糖苷配合,能起到细胞修复作用;转铁蛋白和C-藻蓝素配合,能结合毒性金属离子,减少毒性金属离子抑制脂肪干细胞增殖的作用,提高脂肪干细胞的增殖活性;C-藻蓝素和刺五加皂苷B配合,能够显著增加ERK1/2蛋白的磷酸化水平,从而提高脂肪干细胞的细胞增殖能力;C-藻蓝素,刺五加皂苷B,矢车菊素-3-O-葡萄糖苷和β-蜕皮甾酮配合,能清除过氧化物以及自由基、抑制自由基生成、抑制氧化应激反应及活性代谢物生成等,从而通过多种途径降低自由基和脂质过氧化对脂肪干细胞造成的增殖活性损伤,提高脂肪干细胞的增殖活性。
综上所述,本发明的培养基和培养方法既能促进脂肪干细胞的生长和增殖能力,又能降低培养过程中脂肪干细胞的增殖活性损伤,从而大幅度提高脂肪干细胞的体外扩增效率。
具体实施方式
下面,通过具体实施例对本发明的技术方案进行详细说明。
实施例1
一种无血清脂肪干细胞培养基,包括:DMEM/F12培养基,终浓度为8μg/mL的转铁蛋白,终浓度为30ng/mL的成纤维细胞生长因子,终浓度为2μg/mL的C-藻蓝素,终浓度为0.9μg/mL的刺五加皂苷B,终浓度为0.8μg/mL的矢车菊素-3-O-葡萄糖苷,终浓度为10μg/mL的β-蜕皮甾酮。
实施例2
一种无血清脂肪干细胞培养基,包括:DMEM/F12培养基,终浓度为10μg/mL的转铁蛋白,终浓度为38ng/mL的成纤维细胞生长因子,终浓度为2.8ng/mL的C-藻蓝素,终浓度为1.2μg/mL的刺五加皂苷B,终浓度为1.6μg/mL的矢车菊素-3-O-葡萄糖苷,终浓度为14μg/mL的β-蜕皮甾酮。
实施例3
一种无血清脂肪干细胞培养基,包括:DMEM/F12培养基,终浓度为12μg/mL的转铁蛋白,终浓度为40ng/mL的成纤维细胞生长因子,终浓度为μg/mL的C-藻蓝素,终浓度为1.6μg/mL的刺五加皂苷B,终浓度为2.2μg/mL的矢车菊素-3-O-葡萄糖苷,终浓度为20μg/mL的β-蜕皮甾酮。
实施例4
一种脂肪干细胞的培养方法,具体步骤如下:取大鼠脂肪组织,用PBS清洗后剪碎,加入0.1%的I型胶原酶消化15min,1200r/min离心10min,然后用pH为7.2的D-Hank's液重悬,经过100目筛网过滤,1200r/min离心10min,将获得的细胞按接种密度为1×104/cm2接种于用质量浓度0.2%的明胶溶液包被处理后的培养皿中,加入实施例1中的无血清脂肪干细胞培养基,在37℃、CO2浓度5%条件下进行培养,24h后弃去原培养基,更换新鲜培养基,之后每24h更换培养基一次,待细胞长到80%融合度后,采用浓度为0.25%的胰酶-EDTA溶液消化,按1:3比例传代。
实施例5
一种脂肪干细胞的培养方法,具体步骤如下:取大鼠脂肪组织,用PBS清洗后剪碎,加入0.1%的I型胶原酶消化15min,1200r/min离心10min,然后用pH为7.2的D-Hank's液重悬,经过100目筛网过滤,1200r/min离心10min,将获得的细胞按接种密度为1×104/cm2接种于用质量浓度0.2%的明胶溶液包被处理后的培养皿中,加入实施例2中的无血清脂肪干细胞培养基,在37℃、CO2浓度5%条件下进行培养,24h后弃去原培养基,更换新鲜培养基,之后每24h更换培养基一次,待细胞长到80%融合度后,采用浓度为0.25%的胰酶-EDTA溶液消化,按1:3比例传代。
实施例6
一种脂肪干细胞的培养方法,具体步骤如下:取大鼠脂肪组织,用PBS清洗后剪碎,加入0.1%的I型胶原酶消化15min,1200r/min离心10min,然后用pH为7.2的D-Hank's液重悬,经过100目筛网过滤,1200r/min离心10min,将获得的细胞按接种密度为1×105/cm4接种于用质量浓度0.2%的明胶溶液包被处理后的培养皿中,加入实施例3中的无血清脂肪干细胞培养基,在37℃、CO2浓度5%条件下进行培养,24h后弃去原培养基,更换新鲜培养基,之后每24h更换培养基一次,待细胞长到80%融合度后,采用浓度为0.25%的胰酶-EDTA溶液消化,按1:3比例传代。
对比例1
一种脂肪干细胞的培养方法,具体步骤如下:取大鼠脂肪组织,用PBS清洗后剪碎,加入0.1%的I型胶原酶消化15min,1200r/min离心10min,然后用pH为7.2的D-Hank's液重悬,经过100目筛网过滤,1200r/min离心10min,将获得的细胞按接种密度为1×105/cm4接种于用质量浓度0.2%的明胶溶液包被处理后的培养皿中,加入含10%胎牛血清的DMEM/F12培养基,在37℃、CO2浓度5%条件下进行培养,24h后弃去原培养基,更换新鲜培养基,之后每24h更换培养基一次,待细胞长到80%融合度后,采用浓度为0.25%的胰酶-EDTA溶液消化,按1:3比例传代。
实验例1脂肪干细胞的增殖活性检测
每隔8h,观测实施例4-6以及对比例1中的细胞融合度,并记录细胞长到80%融合度的时间,结果如表1所示:
表1脂肪干细胞的增殖活性
由此可见,本发明的培养基能大幅度提高脂肪干细胞的增殖速度。
实验例2脂肪干细胞的细胞表性检测
按实施例的4-6培养方法培养脂肪干细胞至第5代,收集细胞,用流式细胞仪检测细胞表面抗原的表达水平,其中实施例4-6脂肪干细胞的CD14、CD31、CD45均<1%,呈阴性;CD29、CD73、CD90、CD105均>95%,呈阳性。由此可见,使用本发明培养基以及培养方法培养的脂肪干细胞能保持良好的干细胞特性。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种无血清脂肪干细胞培养基,其特征在于,包括基础培养基和添加剂;所述基础培养基为DMEM/F12培养基,所述添加剂包括:转铁蛋白,成纤维细胞生长因子,C-藻蓝素,刺五加皂苷B,矢车菊素-3-O-葡萄糖苷,β-蜕皮甾酮。
2.根据权利要求1所述的无血清脂肪干细胞培养基,其特征在于,所述添加剂的终浓度为:转铁蛋白8-12μg/mL,成纤维细胞生长因子30-40ng/mL,C-藻蓝素2-3μg/mL,刺五加皂苷B0.9-1.6μg/mL,矢车菊素-3-O-葡萄糖苷0.8-2.2μg/mL,β-蜕皮甾酮10-20μg/mL。
3.根据权利要求1所述的无血清脂肪干细胞培养基,其特征在于,所述添加剂的终浓度为:转铁蛋白10μg/mL,成纤维细胞生长因子38ng/mL,C-藻蓝素2.8μg/mL,刺五加皂苷B1.2μg/mL,矢车菊素-3-O-葡萄糖苷1.6μg/mL,β-蜕皮甾酮14μg/mL。
4.一种脂肪干细胞的培养方法,其特征在于,采用权利要求1-3任一项所述的无血清脂肪干细胞培养基培养。
5.根据权利要求4所述的脂肪干细胞的培养方法,其特征在于,包括以下步骤:
S1、取脂肪组织,用PBS清洗后剪碎,加入0.1-0.15%的I型胶原酶消化15-20min,1200-1500r/min离心10-15min,然后用pH为7.2-7.4的D-Hank's液重悬,经过100-200目筛网过滤,1200-1500r/min离心10-15min;
S2、将步骤S1获得的细胞接种于培养皿中,加入所述无血清脂肪干细胞培养基进行培养,待细胞长到80-90%融合度后消化,按1:3-1:4比例传代。
6.根据权利要求5所述的脂肪干细胞的培养方法,其特征在于,将脂肪干细胞接种于培养皿中,加入所述无血清脂肪干细胞培养基进行培养,24h后弃去原培养基,更换新鲜培养基,之后每24h更换培养基一次,待细胞长到80%融合度后消化,按1:3比例传代。
7.根据权利要求5或6所述的脂肪干细胞的培养方法,其特征在于,所述脂肪干细胞的接种密度为1-1.5×104/cm2。
8.根据权利要求5-7中任一项所述的脂肪干细胞的培养方法,其特征在于,所述培养条件为37℃、CO2浓度5%。
9.根据权利要求5-8中任一项所述的脂肪干细胞的培养方法,其特征在于,所述步骤S2中,消化采用浓度为0.25%的胰酶-EDTA溶液。
10.根据权利要求5-9中任一项所述的脂肪干细胞的培养方法,其特征在于,所述培养皿在接种前,先用促进细胞贴壁的包被液进行包被,优选地,所述包被液为质量浓度0.2-0.3%的明胶溶液。
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