CN115386546A - 一种脐带间充质干细胞的培养方法 - Google Patents
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Abstract
本发明公开了一种脐带间充质干细胞的培养方法,包括以下步骤:(1)自脐带中剥离华通氏胶组织,将华通氏胶组织剪碎后平铺于培养容器中;(2)向步骤(1)的培养容器中加入原代培养基进行培养,待细胞融合度达到80‑90%后用0.25%胰蛋白酶消化传代;(3)将步骤(2)收集的脐带间充质干细胞用传代培养基重悬,进行传代培养。本发明提供的培养方法分别在脐带间充质干细胞的原代和传代培养过程中采用不同成分的培养基。其中在原代培养过程中添加腺苷、瓜籽蛋白、芝麻酚等成分协同作用,有效缩短脐带间充质干细胞原代培养的时间,提高培养效率。在传代培养过程中添加白桦提取物和培养基中的其他成分共同作用,提高脐带间充质干细胞的增殖活性。
Description
技术领域
本发明涉及干细胞领域,尤其涉及一种脐带间充质干细胞的培养方法。
背景技术
间充质干细胞是一类起源于早期中胚层,并且能够自我更新和具有多向分化潜能的成体干细胞,其来源丰富,可从脐带、脂肪、骨髓、牙髓中分离提取。并表现出旁分泌、免疫调节等多种功能,间充质干细胞被认为是细胞治疗最具应用前景的种子细胞。间充质干细胞来源多样、易分离扩增培养、免疫原性低、更新和分化能力强、治疗范围广等。目前间充质干细胞疗法可以作为一种新兴疗法用于治疗骨/软骨疾病、肾病、糖尿病、神经系统疾病以及免疫性疾病等临床领域。
脐带中的干细胞含量较丰富,也最容易获得,脐带间充质干细胞来源于产妇产后的脐带组织,通常情况下是作为医疗废物丢弃的,从脐带组织中分离提取干细胞不存在伦理道德争议,应用前景广阔。近年来,脐带间充质干细胞的体外扩增是在补充有胎牛血清或补充有人的自体血清的培养基中进行。然而,牛血清、人血清或其它动物血清可能含有血液传播的病原体,牛血清还会激发抗异性生物质蛋白抗体的产生,异型生物质蛋白可激发受体患者的免疫应答,另外,牛血清还会显示出批次间差异,导致性能不一致。
现有的一些脐带间充质干细胞在培养过程中也有选择不添加血清的,但是培养效果不理想,存在细胞的增殖速度慢、表达低和获得增殖的细胞数量有限等技术缺陷。因此,如何提高脐带间充质干细胞体外扩增活性是脐带间充质干细胞研究领域的重要问题。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种脐带间充质干细胞的培养方法,该方法可以缩短脐带间充质干细胞原代培养过程的时间,提高脐带间充质干细胞传代培养的细胞增殖活力。
本发明的目的采用如下技术方案实现:
一种脐带间充质干细胞的培养方法,包括以下步骤:
(1)自脐带中剥离华通氏胶组织,将华通氏胶组织剪碎后平铺于培养容器中;
(2)向步骤(1)的培养容器中加入原代培养基进行培养,所述原代培养基由基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺10-20μg/mL、腺苷5-10μg/mL、南瓜籽蛋白20-30ng/mL、芝麻酚10-15ng/mL、维生素C 40-50ng/mL、胰岛素10-15μg/mL,待细胞融合度达到80-90%后用0.25%胰蛋白酶消化传代;
(3)将步骤(2)收集的脐带间充质干细胞用传代培养基重悬,进行传代培养,所述传代培养基的组成为:基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺10-20μg/mL、腺苷5-10μg/mL、白桦提取物1-5μg/mL、维生素C 40-50ng/mL、胰岛素10-15μg/mL。
优选地,步骤(2)和步骤(3)的基础培养基为DMEM/F12。
优选地,所述白桦提取物通过以下方法制备得到:取白桦树皮清洗干净后烘干粉碎,然后以1g:10-15mL的比例加入70-80%乙醇溶液,回流提取2-3h,收集提取液,将提取液减压浓缩后烘干得到白桦提取物。
优选地,步骤(1)中将华氏通胶剪至1-2mm3后平铺于培养容器中。
优选地,步骤(2)中每3-4天更换一次培养基,在37℃,5%CO2的条件下进行原代培养。
优选地,步骤(2)中基础培养基中添加以下成分:L-谷氨酰胺15μg/mL、腺苷7μg/mL、南瓜籽蛋白25ng/mL、芝麻酚13ng/mL、维生素C 45ng/mL、胰岛素11μg/mL。
优选地,步骤(3)中传代培养基中脐带间充质干细胞的密度为1-5×104个/mL。
优选地,步骤(3)传代培养过程中每2-3天更换一次培养基,在37℃,5%CO2的条件下进行传代培养。
优选地,步骤(3)中基础培养基中各成分的浓度为:L-谷氨酰胺15μg/mL、腺苷7μg/mL、白桦提取物4μg/mL、维生素C 45ng/mL、胰岛素13μg/mL。
相比现有技术,本发明的有益效果在于:本发明提供了一种间充质干细胞的培养方法,分别在脐带间充质干细胞的原代和传代培养过程中采用不同成分的培养基。其中在原代培养过程中添加腺苷、瓜籽蛋白、芝麻酚等成分协同作用,有效缩短脐带间充质干细胞原代培养的时间,提高培养效率。在传代培养过程中添加白桦提取物和培养基中的其他成分共同作用,提高脐带间充质干细胞的增殖活性。
具体实施方式
下面,结合具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
一种脐带间充质干细胞的培养方法,其特征在于,包括以下步骤:
(1)自脐带中剥离出华通氏胶组织,用生理盐水清洗3遍后将华通氏胶组织剪至1-2mm3后平铺于培养皿中;
(2)向步骤(1)的培养皿中加入原代培养基,在37℃,5%CO2的条件下进行原代培养,培养过程中每3天更换一次培养基,原代培养基由基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺15μg/mL、腺苷7μg/mL、南瓜籽蛋白25ng/mL、芝麻酚13ng/mL、维生素C 45ng/mL、胰岛素11μg/mL,待细胞融合度达到80-90%后用0.25%胰蛋白酶消化传代;
(3)将步骤(2)收集的脐带间充质干细胞用传代培养基重悬,分装在T75培养瓶中,在37℃,5%CO2的条件下进行传代培养,传代培养基中脐带间充质干细胞的密度为3×104个/mL,传代培养过程中每2天更换一次培养基,传代培养基的组成为:基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺15μg/mL、腺苷7μg/mL、白桦提取物4μg/mL、维生素C 45ng/mL、胰岛素13μg/mL。
白桦提取物通过以下方法制备得到:取白桦树皮清洗干净后烘干粉碎,然后以1g:12mL的比例加入75%乙醇溶液,回流提取2.5h,收集提取液,将提取液减压浓缩后烘干得到白桦提取物。
实施例2
一种脐带间充质干细胞的培养方法,其特征在于,包括以下步骤:
(1)自脐带中剥离出华通氏胶组织,用生理盐水清洗3遍后将华通氏胶组织剪至1-2mm3后平铺于培养皿中;
(2)向步骤(1)的培养皿中加入原代培养基,在37℃,5%CO2的条件下进行原代培养,培养过程中每4天更换一次培养基,原代培养基由基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺10μg/mL、腺苷5μg/mL、南瓜籽蛋白20ng/mL、芝麻酚10ng/mL、维生素C 40ng/mL、胰岛素10μg/mL,待细胞融合度达到80%后用0.25%胰蛋白酶消化传代;
(3)将步骤(2)收集的脐带间充质干细胞用传代培养基重悬,分装在T75培养瓶中,在37℃,5%CO2的条件下进行传代培养,传代培养基中脐带间充质干细胞的密度为1×104个/mL,传代培养过程中每2天更换一次培养基,传代培养基的组成为:基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺10μg/mL、腺苷5μg/mL、白桦提取物1μg/mL、维生素C 40ng/mL、胰岛素10μg/mL。
白桦提取物通过以下方法制备得到:取白桦树皮清洗干净后烘干粉碎,然后以1g:10mL的比例加入70%乙醇溶液,回流提取3h,收集提取液,将提取液减压浓缩后烘干得到白桦提取物。
实施例3
一种脐带间充质干细胞的培养方法,其特征在于,包括以下步骤:
(1)自脐带中剥离出华通氏胶组织,用生理盐水清洗3遍后将华通氏胶组织剪至1-2mm3后平铺于培养皿中;
(2)向步骤(1)的培养皿中加入原代培养基,在37℃,5%CO2的条件下进行原代培养,培养过程中每3天更换一次培养基,原代培养基由基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺20μg/mL、腺苷10μg/mL、南瓜籽蛋白30ng/mL、芝麻酚15ng/mL、维生素C 50ng/mL、胰岛素15μg/mL,待细胞融合度达到90%后用0.25%胰蛋白酶消化传代;
(3)将步骤(2)收集的脐带间充质干细胞用传代培养基重悬,分装在T75培养瓶中,在37℃,5%CO2的条件下进行传代培养,传代培养基中脐带间充质干细胞的密度为5×104个/mL,传代培养过程中每3天更换一次培养基,传代培养基的组成为:基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺20μg/mL、腺苷10μg/mL、白桦提取物5μg/mL、维生素C 50ng/mL、胰岛素15μg/mL。
白桦提取物通过以下方法制备得到:取白桦树皮清洗干净后烘干粉碎,然后以1g:15mL的比例加入80%乙醇溶液,回流提取3h,收集提取液,将提取液减压浓缩后烘干得到白桦提取物。
对比例1
对比例1提供一种脐带间充质干细胞的培养方法,和实施例1的区别为:省去南瓜籽蛋白,其余均和实施例1相同。
对比例2
对比例2提供一种脐带间充质干细胞的培养方法,和实施例1的区别为:省去芝麻酚,其余均和实施例1相同。
对比例3
对比例3提供一种脐带间充质干细胞的培养方法,和实施例1的区别为:省去芝麻酚,将维生素C的用量调整为58ng/mL,其余均和实施例1相同。
对比例4
对比例4提供一种脐带间充质干细胞的培养方法,和实施例1的区别为:省去白桦提取物,其余均和实施例1相同。
对比例5
对比例5提供一种脐带间充质干细胞的培养方法,和实施例1的区别为:将步骤(3)的白桦提取物省去,调整为在步骤(2)中的原代培养基中添加白桦提取物,用量为4μg/mL,其余均和实施例1相同。
对比例6
对比例6提供一种脐带间充质干细胞的培养方法,和实施例1的区别为:将步骤(2)的南瓜籽蛋白、芝麻酚省去,调整为在步骤(3)中的传代培养基中添加南瓜籽蛋白25ng/mL、芝麻酚13ng/mL,其余均和实施例1相同。
分别统计实施例1至3,对比例1至6中脐带间充质干细胞原代培养过程所用时间,即细胞融合度达到80-90%所用的培养时间,结果如表1所示。
表1
组别 | 时间(d) |
实施例1 | 9 |
实施例2 | 11 |
实施例3 | 10 |
对比例1 | 14 |
对比例2 | 15 |
对比例3 | 14 |
对比例4 | 9 |
对比例5 | 9 |
对比例6 | 20 |
由表1可以看出,实施例1至3中的脐带间充质干细胞所需的原代培养时间较短,对比例1至6中调整了培养过程中各组分的添加时机,细胞的原代培养时间变长。说明采用本发明的培养方法有助于缩短脐带间充质干细胞原代培养时间,加快了脐带间充质干细胞自组织中爬出的速度,缩短培养周期,降低培养成本。
采用台盼蓝染色方分别统计实施例1,对比例1至6中的脐带间充质干细胞在传代培养7天后,脐带间充质干细胞的增殖倍数,计算方式如下:增殖倍数=传代培养7d后收集的细胞总数/用于传代培养的初始细胞接种量,结果如表2所示。
表2
由表2可以看出,实施例1中用于传代的脐带间充质干细胞的增殖能力最好,在短时间内可培养得到大量的脐带间充质干细胞。对比例1至6中分别调整了培养过程中部分成分的添加时机,除对比例6外细胞的增殖能力均有不同程度的下降,说明采用本发明的培养方法,有效提高脐带间充质干细胞的增殖活性,提高细胞体外扩增的效率。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (9)
1.一种脐带间充质干细胞的培养方法,其特征在于,包括以下步骤:
(1)自脐带中剥离华通氏胶组织,将华通氏胶组织剪碎后平铺于培养容器中;
(2)向步骤(1)的培养容器中加入原代培养基进行培养,所述原代培养基由基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺10-20μg/mL、腺苷5-10μg/mL、南瓜籽蛋白20-30ng/mL、芝麻酚10-15ng/mL、维生素C 40-50ng/mL、胰岛素10-15μg/mL,待细胞融合度达到80-90%后用0.25%胰蛋白酶消化传代;
(3)将步骤(2)收集的脐带间充质干细胞用传代培养基重悬,进行传代培养,所述传代培养基的组成为:基础培养基以及添加在基础培养基中的以下成分:L-谷氨酰胺10-20μg/mL、腺苷5-10μg/mL、白桦提取物1-5μg/mL、维生素C 40-50ng/mL、胰岛素10-15μg/mL。
2.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(2)和步骤(3)的基础培养基为DMEM/F12。
3.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,所述白桦提取物通过以下方法制备得到:取白桦树皮清洗干净后烘干粉碎,然后以1g:10-15mL的比例加入70-80%乙醇溶液,回流提取2-3h,收集提取液,将提取液减压浓缩后烘干得到白桦提取物。
4.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(1)中将华氏通胶剪至1-2mm3后平铺于培养容器中。
5.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(2)中每3-4天更换一次培养基,在37℃,5%CO2的条件下进行原代培养。
6.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(2)中基础培养基中添加以下成分:L-谷氨酰胺15μg/mL、腺苷7μg/mL、南瓜籽蛋白25ng/mL、芝麻酚13ng/mL、维生素C 45ng/mL、胰岛素11μg/mL。
7.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(3)中传代培养基中脐带间充质干细胞的密度为1-5×104个/mL。
8.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(3)传代培养过程中每2-3天更换一次培养基,在37℃,5%CO2的条件下进行传代培养。
9.根据权利要求1所述脐带间充质干细胞的培养方法,其特征在于,步骤(3)中基础培养基中各成分的浓度为:L-谷氨酰胺15μg/mL、腺苷7μg/mL、白桦提取物4μg/mL、维生素C45ng/mL、胰岛素13μg/mL。
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