CN114561347A - 一种脂肪间充质干细胞的培养基和培养方法 - Google Patents
一种脂肪间充质干细胞的培养基和培养方法 Download PDFInfo
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Abstract
本申请涉及干细胞培养技术领域,具体公开了一种脂肪间充质干细胞的培养基和培养方法。所述培养基包括基础培养基和血清替代物;血清替代物包括以下组分:人血清白蛋白、转铁蛋白、纤粘连蛋白、层粘连蛋白、雌二醇、辅酶A、牛磺酸、丙酮酸、血管内皮生长因子、血小板衍生生长因子、碱性成纤维细胞生长因子、转化生长因子‑α、转化生长因子‑β、L‑谷氨酰胺、胰岛素样生长因子‑I、白细胞介素‑21、胰酶抑制剂、蛋白酶抑制剂、人重组蛋白PDCD5、藁本内酯、川芎内酯、地黄苷、黄芩素、芍药苷。所述培养方法包括以下步骤:将原代或传代细胞重悬在培养基中得到脂肪间充质干细胞重悬液,培养。本申请提高体外脂肪间充质干细胞的增殖能力。
Description
技术领域
本申请涉及干细胞培养技术领域,具体涉及一种脂肪间充质干细胞的培养基和培养方法。
背景技术
脂肪间充质干细胞具有强大的自我更新能力和多向分化潜能,是移植范畴使用研究远景最大的再生来源干细胞。人脂肪间充质干细胞在不同的条件下被成功诱导分化为:成骨细胞、软骨细胞、骨骼肌细胞、肝脏细胞等中胚层细胞,同时也可以向其他胚层分化,间充质干细胞表面分子及特征与其他组织来源的间充质干细胞相似。
脂肪间充质干细胞已在治疗慢性疾病、自身免疫疾病及自身运动损伤方面取得了很大的进展。脂肪间充质干细胞应用于临床治疗和再生医学的潜能已被干细胞研究者们广泛关注。而一个治疗的疗程至少需要1×107个细胞,但从患者、捐赠者身上无法获得足够的脂肪间充质干细胞,限制了其应用。
因此,探索通过改变各种培养条件,促进体内来源的脂肪间充质干细胞的体外增殖,使体外脂肪间充质干细胞快速增殖成为亟需。
发明内容
为了提高体外脂肪间充质干细胞的增殖能力,本申请提供一种脂肪间充质干细胞的培养基和培养方法。
第一方面,本申请提供的一种脂肪间充质干细胞的培养基,采用如下技术方案:
一种脂肪间充质干细胞的培养基,所述培养基包括基础培养基和血清替代物;
所述血清替代物包括以下组分,且各组分在所述培养基中的终浓度为:
人血清白蛋白:1~2g/L、转铁蛋白:5~15mg/L、纤粘连蛋白:3~7mg/L、层粘连蛋白:2~5mg/L、雌二醇:1~5μg/L、辅酶A:100~200mg/L、牛磺酸:2~3mg/L、丙酮酸:8~28mg/L、血管内皮生长因子:4~10μg/L、血小板衍生生长因子:120~150μg/L、碱性成纤维细胞生长因子:5~15μg/L、转化生长因子-α:1~4μg/L、转化生长因子-β:1~4μg/L、L-谷氨酰胺:300~500mg/L、胰岛素样生长因子-I:5~8μg/L、白细胞介素-21:1.5~3.5μg/L、胰酶抑制剂:0.3~0.8mg/L、蛋白酶抑制剂:2~3.6μg/L、人重组蛋白PDCD5:6~10mg/L、藁本内酯:2~5μg/L、川芎内酯:2~5g/L、地黄苷:2~5μg/L、黄芩素:2~5μg/L、芍药苷:2~5μg/L。
可选的,所述血清替代物还包括抗炎抗敏物质,且所述抗炎抗敏物质在所述培养基中的终浓度为0.36~1.09mg/L。
可选的,所述抗炎抗敏物质选自地塞米松和氢化可的松中的至少一种。
可选的,所述基础培养基为HYStemCell人间充质干细胞无血清培养基。
第二方面,本申请提供的一种脂肪间充质干细胞的培养方法,采用如下技术方案:
一种脂肪间充质干细胞的培养方法,所述培养方法包括以下步骤:
将脂肪间充质干细胞的原代或传代细胞重悬在如权利要求1至4中任意一项所述培养基中得到脂肪间充质干细胞重悬液,培养脂肪间充质干细胞重悬液,得到脂肪间充质干细胞。
可选的,所述脂肪间充质干细胞重悬液的浓度为(1~2)×107个/mL。
可选的,所述培养的条件是:在温度为37℃、体积分数为5%的二氧化碳培养箱中培养。
综上所述,本申请具有以下有益效果:
第一、本申请的培养基体外培养脂肪间充质干细胞,增殖能力强。
第二、采用本申请的培养基培养所得到的脂肪间充质干细胞纯度高、质量好。
具体实施方式
以下结合实施例对本申请作进一步详细说明。
脂肪间充质干细胞的培养基的制备
制备例1
制备例1提供一种脂肪间充质干细胞的培养基包括基础培养基和血清替代物;
基础培养基为HYStemCell人间充质干细胞无血清培养基;
血清替代物包括以下组分,且各组分在所述培养基中的终浓度为:
人血清白蛋白:1.3g/L、转铁蛋白:10mg/L、纤粘连蛋白:5mg/L、层粘连蛋白:4mg/L、雌二醇:3μg/L、辅酶A:160mg/L、牛磺酸:2.5mg/L、丙酮酸:18mg/L、血管内皮生长因子:7μg/L、血小板衍生生长因子:135μg/L、碱性成纤维细胞生长因子:10μg/L、转化生长因子-α:2.5μg/L、转化生长因子-β:2.5μg/L、L-谷氨酰胺:400mg/L、胰岛素样生长因子-I:6.5μg/L、白细胞介素-21:2.5μg/L、胰酶抑制剂:0.55mg/L、蛋白酶抑制剂:2.8μg/L、人重组蛋白PDCD5:8mg/L、藁本内酯:3.5μg/L、川芎内酯:3.5g/L、地黄苷:3.5μg/L、黄芩素:3.5μg/L、芍药苷:3.5μg/L、地塞米松0.36mg/L。
制备对比例1
与制备例1相比,不同的是,制备对比例1未添加人重组蛋白PDCD5。
制备对比例2
与制备例1相比,不同的是,制备对比例2未添加藁本内酯。
制备对比例3
与制备例1相比,不同的是,制备对比例3未添加川芎内酯。
制备对比例4
与制备例1相比,不同的是,制备对比例4未添加地黄苷。
制备对比例5
与制备例1相比,不同的是,制备对比例5未添加黄芩素。
制备对比例6
与制备例1相比,不同的是,制备对比例6未添加芍药苷。
脂肪间充质干细胞的原代细胞的获得
在超净工作台内,使用含青链霉素(青霉素500units/mL、链霉素500μg/mL)的PBS缓冲液反复冲洗人脂肪组织至无血色,去除人脂肪组织内结缔组织和血管,最后剪成约1mm3大小的碎块。
向碎块状的人脂肪组织中加入质量百分比浓度为0.25%的胰蛋白酶细胞消化液,37℃恒温震荡消化45~60min,至人脂肪组织呈糊状;加入质量百分比浓度为10%的胎牛血清的培养液终止消化,然后用100目筛网过滤去除未消化的人脂肪组织,1200r/min离心10min,弃去上层的脂滴及中间层的培养基,得到下层的脂肪间充质干细胞的原代细胞。
脂肪间充质干细胞的培养
脂肪间充质干细胞的培养方法,包括以下步骤:
将脂肪间充质干细胞的原代细胞用培养基重悬得到浓度为1×107个/mL的脂肪间充质干细胞重悬液,将脂肪间充质干细胞重悬液按3×103个/cm2接种于24孔板;之后将24孔板置于温度为37℃、体积分数为5%的二氧化碳培养箱中培养7d。
实施例1和对比例1~6
实施例1和对比例1~6的区别仅在于,所使用的培养基不同;其中,实施例1和对比例1~6所使用培养基如表1所示。
表1实施例1和对比例1~6所使用培养基
项目 | 培养基 |
实施例1 | 制备例1 |
对比例1 | 制备对比例1 |
对比例2 | 制备对比例2 |
对比例3 | 制备对比例3 |
对比例4 | 制备对比例4 |
对比例5 | 制备对比例5 |
对比例6 | 制备对比例6 |
培养方法的增殖能力检测
检测方法:取3个孔,每个孔分别采用台盼蓝拒染法计数活细胞数,计算3个孔的活细胞数平均值。实施例1和对比例1~6的活细胞数平均值如表2所示。
表2实施例1和对比例1~6的活细胞数平均值
项目 | 活细胞数平均值 |
实施例1 | 2.0×10<sup>4</sup>个 |
对比例1 | 1.2×10<sup>4</sup>个 |
对比例2 | 1.3×10<sup>4</sup>个 |
对比例3 | 1.4×10<sup>4</sup>个 |
对比例4 | 1.7×10<sup>4</sup>个 |
对比例5 | 1.5×10<sup>4</sup>个 |
对比例6 | 1.5×10<sup>4</sup>个 |
从表2可以看出,采用制备例1的培养基对脂肪间充质干细胞进行培养,脂肪间充质干细胞的增殖能力得到显著提高。
脂肪间充质干细胞的免疫表型检测
免疫表型检测方法为:分别向实施例1和对比例1~6得到的脂肪间充质干细胞中加入质量百分比浓度为0.25%的胰蛋白酶细胞消化液,取1×105个细胞,加入免疫表型标志物(例如:CD29、CD73、CD90、CD105、CD14、CD34、CD45、HLA-DR抗体)并混匀,避光孵育30min;检测脂肪间充质干细胞的免疫表型表达,检测结果如表3所示。
表3实施例1和对比例1~6脂肪间充质干细胞的免疫表型表达结果
从表3可以看出,实施例1所得到的脂肪间充质干细胞的阳性标志物表达明显高于对比例1~6,而实施例1所得到的脂肪间充质干细胞的阴性标志物表达明显低于对比例1~6。说明采用本申请的培养基培养所得到的脂肪间充质干细胞纯度高、质量好。
可以理解的是,以上实施方式仅仅是为了说明本发明的原理而采用的示例性实施方式,然而本发明并不局限于此。对于本领域内的普通技术人员而言,在不脱离本发明的精神和实质的情况下,可以做出各种变型和改进,这些变型和改进也视为本发明的保护范围。
Claims (7)
1.一种脂肪间充质干细胞的培养基,其特征在于,所述培养基包括基础培养基和血清替代物;
所述血清替代物包括以下组分,且各组分在所述培养基中的终浓度为:
人血清白蛋白:1~2g/L、转铁蛋白:5~15mg/L、纤粘连蛋白:3~7mg/L、层粘连蛋白:2~5mg/L、雌二醇:1~5μg/L、辅酶A:100~200mg/L、牛磺酸:2~3mg/L、丙酮酸:8~28mg/L、血管内皮生长因子:4~10μg/L、血小板衍生生长因子:120~150μg/L、碱性成纤维细胞生长因子:5~15μg/L、转化生长因子-α:1~4μg/L、转化生长因子-β:1~4μg/L、L-谷氨酰胺:300~500mg/L、胰岛素样生长因子-I:5~8μg/L、白细胞介素-21:1.5~3.5μg/L、胰酶抑制剂:0.3~0.8mg/L、蛋白酶抑制剂:2~3.6μg/L、人重组蛋白PDCD5:6~10mg/L、藁本内酯:2~5μg/L、川芎内酯:2~5g/L、地黄苷:2~5μg/L、黄芩素:2~5μg/L、芍药苷:2~5μg/L。
2.根据权利要求1所述的培养基,其特征在于,所述血清替代物还包括抗炎抗敏物质,且所述抗炎抗敏物质在所述培养基中的终浓度为0.36~1.09mg/L。
3.根据权利要求2所述的培养基,其特征在于,所述抗炎抗敏物质选自地塞米松和氢化可的松中的至少一种。
4.根据权利要求1所述的培养基,其特征在于,所述基础培养基为HYStemCell人间充质干细胞无血清培养基。
5.一种脂肪间充质干细胞的培养方法,其特征在于,所述培养方法包括以下步骤:
将脂肪间充质干细胞的原代或传代细胞重悬在如权利要求1至4中任意一项所述培养基中得到脂肪间充质干细胞重悬液,培养脂肪间充质干细胞重悬液,得到脂肪间充质干细胞。
6.根据权利要求5所述的培养方法,其特征在于,所述脂肪间充质干细胞重悬液的浓度为(1~2)×107个/mL。
7.根据权利要求5所述的培养方法,其特征在于,所述培养的条件是:在温度为37℃、体积分数为5%的二氧化碳培养箱中培养。
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