CN111471651A - 一种人脂肪干细胞无血清培养基及其制备方法 - Google Patents
一种人脂肪干细胞无血清培养基及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种人脂肪干细胞无血清培养基,包括DMEM/F12基础培养基和添加成分,其中添加成分为:人血清白蛋白、B27添加剂或类似物、碱性成纤维生长因子、酸性成纤维生长因子、血小板源生长因子、表皮生长因子、胰岛素样生长因子、肝素、纤粘连蛋白、抗坏血酸磷酸酯镁盐、N‑乙酰半胱氨酸、庆大霉素和两性霉素。本发明人脂肪干细胞无血清培养基,不含任何血清成分,且成分明确,避免了动物血清内潜在动物源性内毒素或病毒将对人体健康构成极大地风险,所含组分均来自重组蛋白或化学合成,不会引起人体免疫排斥,解决了应用的安全性问题。
Description
技术领域
本发明属于细胞培养技术领域,具体地说,涉及一种人脂肪干细胞无血清培养基及其制备方法。
背景技术
目前常用的人脂肪干细胞(hADSCs)培养是利用饲养层细胞与采用含10%胎牛血清的培养基进行培养。这种培养干细胞方法的缺点是方法复杂,提取干细胞数量少,纯度不高,继代增殖缓慢,更大问题是使用饲养层细胞和动物血清容易导致干细胞污染,特别是动物血清内潜在动物源性内毒素或病毒将对人体健康构成极大地风险,这样培养出的干细胞不适于直接应用于临床。因此,开发一种合适的有效的培养基变的迫在眉睫。
发明内容
本发明的目的是提供一种人脂肪干细胞无血清培养基,其不含动物血清成分,可以完全实现无血清培养人脂肪干细胞;采用本发明无血清培养基培养人脂肪干细胞,可在体外获得高数量、高纯度并且安全优质的人脂肪干细胞,同时可提高细胞存活率,维持干细胞的特性。
本发明的另一个目的是提供一种人脂肪干细胞无血清培养基的制备方法。
为了解决上述技术问题,本发明公开的第一个技术方案是,一种人脂肪干细胞无血清培养基,包括DMEM/F12基础培养基和添加成分,其中添加成分为:HSA、B27、bFGF、aFGF、PDGF、EGF、HPS、FN、Vc-Mg、NAC、 Gent-AMP。
进一步地,DMEM/F12基础培养基中DMEM培养基与F12培养基的质量比为1:2。
优选地,添加成分在DMEM/F12基础培养基中的含量为:
B27添加剂或类似物:0.5-2×;
HSA:2-50mg/ml;
IGF:1-100ng/ml
bFGF:2-20ng/ml;
aFGF:2-20ng/ml;
PDGF:2-20ng/ml;
EGF:2-20ng/ml;
Vc-Mg:10-100μg/ml
NAC:1-10mg/ml;
HPS:1-10mg/ml;
FN:10-100ug/ml;
庆大霉素:1-20μg/ml;
两性霉素:1-10μg/ml;
更优选地,添加成分在DMEM/F12基础培养基中的含量为:
HSA:5-25mg/ml;
IGF:2-50ng/ml
bFGF:4-10ng/ml;
aFGF:4-10ng/ml;
PDGF:4-10ng/ml;
EGF:4-10ng/ml;
Vc-Mg:20-50μg/ml
NAC:2-10mg/ml;
HPS:2-10mg/ml;
FN:20-50ug/ml;
庆大霉素:1-20μg/ml;
两性霉素:2-10μg/ml;
最优选地,添加成分在DMEM/F12基础培养基中的含量为:
HSA:10mg/ml;
IGF:10ng/ml
bFGF:5ng/ml;
aFGF:5ng/ml;
PDGF:5ng/ml;
EGF:5ng/ml;
Vc-Mg:25μg/ml
NAC:5mg/ml;
HPS:5mg/ml;
FN:25ug/ml;
庆大霉素:10μg/ml;
两性霉素:5μg/ml;
本发明公开的第二个技术方案是,一种人脂肪干细胞无血清培养基的制备方法,在无菌环境下下,取DMEM/F12基础培养基,依次加入HSA、B27、 bFGF、aFGF、PDGF、EGF、HPS、FN、Vc-Mg、NAC、Gent-AMP混合均匀,得到人脂肪干细胞无血清培养基。
本发明公开的第三个技术方案是,人脂肪干细胞无血清培养基在培养人脂肪干细胞中的应用。
与现有技术相比,本发明可以获得包括以下技术效果:
1)本发明人脂肪干细胞无血清培养基,不含任何血清成分,且成分明确,避免了动物血清内潜在动物源性内毒素或病毒将对人体健康构成极大地风险,所含组分均来自重组蛋白或化学合成,不会引起人体免疫排斥,解决了应用的安全性问题。
2)采用本发明无血清培养基培养人脂肪干细胞,细胞增殖速率高,细胞能够保持较高的完整性和活性,适用于大规模的人脂肪干细胞培养,成本低廉且方便配制。
3)采用本发明无血清培养基培养人脂肪干细胞,可在体外获得高数量、高纯度并且安全优质的人脂肪干细胞,同时可提高细胞存活率,维持干细胞的特性。
附图说明
图1是脂肪干细胞在本发明无血清培养基培养下的增殖活性;
图2是脂肪干细胞的流式细胞表性检测。
具体实施方式
以下将配合实施例来详细说明本发明的实施方式,藉此对本发明如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
本发明公开了一种人脂肪干细胞无血清培养基,包括DMEM/F12基础培养基和添加成分,其中添加成分为:HSA、B27、bFGF、aFGF、PDGF、 EGF、HPS、FN、Vc-Mg、NAC、Gent-AMP。
DMEM/F12基础培养基中DMEM培养基与F12培养基的质量比为1:2。
添加成分在DMEM/F12基础培养基中的含量为:
B27添加剂(100×):0.25-2%;
HSA:2-50mg/ml;
IGF:1-100ng/ml
bFGF:2-20ng/ml;
aFGF:2-20ng/ml;
PDGF:2-20ng/ml;
EGF:2-20ng/ml;
Vc-Mg:10-100μg/ml
NAC:1-10mg/ml;
肝素(HPS):1-10mg/ml;
纤粘连蛋白:10-100ug/ml;
庆大霉素:1-20μg/ml;
两性霉素:1-10μg/ml;
本发明还公开了上述人脂肪干细胞无血清培养基的制备方法,在无菌环境下下,取DMEM/F12基础培养基,依次加入人血清白蛋白、B27、转铁蛋白、还原型谷胱甘肽、bFGF、aFGF、PDGF、EGF、Gent-AMP、Vc-Mg和 NAC,混合均匀,得到人脂肪干细胞无血清培养基。
本发明人脂肪干细胞无血清培养基中各组分的作用:
DMEM/F12基础培养基:DMEM培养基是一种含各种氨基酸和葡萄糖的培养基,F12培养基,成分复杂,含有多种微量元素,本发明采用DMEM/F12基础培养基,以利用F12含有较丰富的成份和DMEM含有较高浓度的营养成分的优点。
人血清白蛋白(HSA):能够运输维生素、脂肪酸类等营养物质,促进细胞新陈代谢以及保护细胞不受蛋白酶的损伤。
B27及类似物、是指B27添加剂及其同类产品,还包括B27添加剂的后续改进或替代产品。本方案B27添加剂等可以是由赛默飞世尔(Thermo Fisher)等公司出售的产品,也可以是其他公司出售或按已公开配方配制添加剂,这类产品多用于胚胎干细胞,多能干细胞,神经干细胞及神经元等的培养。本发明的实施例中,采用的是由赛默飞世尔公司出售的B27添加剂。
还原型谷胱甘肽:作为抗氧化剂,具有抑制细胞糖酵解作用,抑制细胞凋亡作用。
L-谷氨酰胺:作为培养细胞的能量来源、参与蛋白质的合成和核酸代谢。
重组碱性成纤维细胞生长因子bFGF、重组酸性性成纤维细胞生长因子 aFGF、血小板衍生因子PDGF和重组表皮生长因子EGF,是维持细胞体外生长增殖、分裂分化所必需的生物补充因子
Vc-Mg:维生素C为脂肪干细胞培养提供营养,参与细胞的蛋白质代谢、脂肪代谢、糖代谢等重要生命活动。
NAC:抗氧化剂
庆大霉素:广谱抗菌剂,保存时间长,在常温性质稳定;
两性霉素:真菌抗菌剂;
下面结合实施例对本发明作进一步的说明。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1制备人脂肪干细胞培养基
包括DMEM/F12基础培养基和添加成分,其中DMEM/F12基础培养基中DMEM培养基与F12培养基的质量比为1:2;
添加成分在DMEM/F12基础培养基中的含量为:
HSA:10mg/ml;
B27或类似物:1%
IGF:10ng/ml
bFGF:5ng/ml;
aFGF:5ng/ml;
PDGF:5ng/ml;
EGF:5ng/ml;
Vc-Mg:25μg/ml
NAC:5mg/ml;
肝素(HPS):5mg/ml;
纤粘连蛋白:25ug/ml;
庆大霉素:10μg/ml;
两性霉素:5μg/ml;
制备方法为:在无菌环境下下,取DMEM/F12基础培养基,依次加入人血清白蛋白、B27、转铁蛋白、还原型谷胱甘肽、bFGF、aFGF、PDGF、EGF、Gent-AMP、Vc-Mg和NAC,混合均匀,即得。
实施例2制备人脂肪干细胞培养基
包括DMEM/F12基础培养基和添加成分,其中DMEM/F12基础培养基中DMEM培养基与F12培养基的质量比为1:2;
添加成分在DMEM/F12基础培养基中的含量为:
HSA:50mg/ml;
B27或类似物:2%
IGF:20ng/ml
bFGF:10ng/ml;
aFGF:10ng/ml;
PDGF:10ng/ml;
EGF:10ng/ml;
Vc-Mg:50μg/ml
NAC:10mg/ml;
HPS:10mg/ml;
纤粘连蛋白:50ug/ml;
庆大霉素:10μg/ml;
两性霉素:5μg/ml;
制备方法为:在无菌环境下下,取DMEM/F12基础培养基,依次加入人血清白蛋白、B27、转铁蛋白、还原型谷胱甘肽、bFGF、aFGF、PDGF、 EGF、Gent-AMP、Vc-Mg和NAC,混合均匀,即得。
实施例3制备人脂肪干细胞培养基
包括DMEM/F12基础培养基和添加成分,其中DMEM/F12基础培养基中DMEM培养基与F12培养基的质量比为1:2;
添加成分在DMEM/F12基础培养基中的含量为:
HSA:5mg/ml;
B27或类似物:0.25%
IGF:5ng/ml
bFGF:2ng/ml;
aFGF:2ng/ml;
PDGF:5ng/ml;
EGF:5ng/ml;
Vc-Mg:10μg/ml
NAC:5mg/ml;
肝素(HPS):5mg/ml;
纤粘连蛋白:20ug/ml;
庆大霉素:10μg/ml;
两性霉素:5μg/ml;
制备方法为:在无菌环境下下,取DMEM/F12基础培养基,依次加入人血清白蛋白、B27、转铁蛋白、还原型谷胱甘肽、bFGF、aFGF、PDGF、 EGF、Gent-AMP、Vc-Mg和NAC,混合均匀,即得。
对比实验
将实施例2制备的无血清培养基置于T25细胞培养瓶中,将人脂肪干细胞以1.0×104cells/cm2接种,在温度37℃下培养,统计原代细胞的长出时间,每代的生长时间。
同时,以Lonza间充质干细胞无血清培养基(市售)为对照培养人脂肪干细胞。
结论:
原代细胞收获时间:通常的细胞培养基进行酶消化法分离细胞,7-9天可收获原代细胞,而本发明的无血清培养基爬出细胞时间缩短至6-7天,提高了效率。
细胞每代生长时间,经过连续10代监测,平均每代生长时间在2-3天,比Lonza无血清培养基培养时间缩短了一天,节约了培养时间,提高了工作效率。
连续传代次数:传统的MSC无血清培养基传代至6代以后,细胞生长变缓慢,本发明无血清培养基可连续传代至10代,细胞仍保持其分化潜能,而对照培养基传代至第9代后,细胞生长明显变缓,导致后期无法传代。
如图1所示,是脂肪干细胞在本发明无血清培养基和含10%血清培养基培养下的增殖活性曲线。由图1所示,实验表明本发明的培养基与含血清培养基无明显差异。
图2是脂肪干细胞的流式细胞表性检测。由图2所见,按本发明的培养方法培养脂肪干细胞至第3代,收集细胞,用流式细胞仪检测细胞表面抗原的表达水平发现CD34、CD45均<5%,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。
上述说明示出并描述了发明的若干优选实施例,但如前所述,应当理解发明并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文所述发明构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离发明的精神和范围,则都应在发明所附权利要求的保护范围内。
Claims (5)
1.一种人脂肪干细胞无血清培养基,其特征在于,包括DMEM/F12基础培养基和添加成分,其中主要添加成分为:人血清白蛋白(HSA)、B27添加剂或类似物、碱性成纤维生长因子(bFGF)、酸性成纤维生长因子(aFGF)、血小板源生长因子(PDGF)、表皮生长因子(EGF)、胰岛素样生长因子(IGF)、肝素(HPS)、纤粘连蛋白、抗坏血酸磷酸酯镁盐(Vc-Mg)、N-乙酰半胱氨酸(NAC)、庆大霉素和两性霉素(Gent-AMP)。
2.根据权要求1所述的人脂肪干细胞无血清培养基,其特征在于,所述DMEM/F12基础培养基中DMEM培养基与F12培养基的质量比为1:2。
3.根据权利要求1或2所述的人脂肪干细胞无血清培养基,其特征在于,所述添加成分在DMEM/F12基础培养基中的含量为:
B27添加剂或类似物(B27)(100×):1×;
HSA:2-50mg/ml;
IGF:1-100ng/ml
bFGF:2-20ng/ml;
aFGF:2-20ng/ml;
PDGF:2-20ng/ml;
EGF:2-20ng/ml;
Vc-Mg:10-100μg/ml
NAC:1-10mg/ml;
肝素(HPS):1-10mg/ml;
纤粘连蛋白(FN):10-100ug/ml;
庆大霉素(Gent):1-20μg/ml;
两性霉素(AMP):1-5μg/ml。
4.权利要求1-3任一项所述的人脂肪干细胞无血清培养基的制备方法,其特征在于,在无菌环境下下,取DMEM/F12基础培养基,依次加入HSA、B27、bFGF、aFGF、PDGF、EGF、HPS、FN、Vc-Mg、NAC、Gent-AMP,混合均匀,得到人脂肪干细胞无血清培养基。
5.权利要求1-4任一项所述的人脂肪干细胞无血清培养基在培养人脂肪干细胞中的应用。
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