CN110628706B - 一种体外提取并培养胚胎神经干细胞的方法及培养基的制备 - Google Patents
一种体外提取并培养胚胎神经干细胞的方法及培养基的制备 Download PDFInfo
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Abstract
本发明公开了一种体外提取培养胚胎神经干细胞的方法,取出生24h内的新生鼠,脱颈处死后,浸入乙醇消毒,氯化钠溶液冲洗,用无菌剪刀于上颈椎和骶椎处剪断脊柱,取出脊柱干部;用宽嘴镊子将脊柱内的脊髓挤出,放入氯化钠溶液中,显微镜下剥离脊膜,将脊髓过滤后放入离心管中;加入胰蛋白酶边吹打边消化,加入DMEM/F12培养基中止消化,细胞滤网过滤后离心,收集细胞,加入胚胎神经干细胞完全培养基进行培养。胚胎神经干细胞完全培养基配方包括DMEM/F12、谷氨酰胺、赖氨酸、B‑27添加物、N2添加物、青霉素、链霉素、bFGF、EGF、胰岛素、小分子抑制剂CHIR99021。
Description
技术领域
本发明属于胚胎神经干细胞技术领域,具体涉及一种体外提取并培养胚胎神经干细胞的方法及培养基的制备。
背景技术
胚胎神经干细胞因其具有多能分化潜能而被认为是修复神经系统损伤的新技术。但是,如何有效提取大量高纯度和高活力的胚胎神经干细胞并进行体外培养,维持其细胞活性和多能分化潜能一直是一个难题。
目前,现有技术提取神经干细胞多提取幼鼠大脑海马区的神经干细胞,可导致提取的神经干细胞中混有大量其他细胞以及血管、脑膜等非细胞成分,导致所提取的神经干细胞纯度较低,不利于后期的细胞培养及实验研究;此外,目前常用的体外培养神经干细胞的培养基多为商品化的培养基,不能针对胚胎神经干细胞的细胞学生物特性进行优化,导致体外培养的胚胎神经干细胞活力较差、细胞增殖缓慢、发生分化甚至大量死亡。
发明内容
本发明的目的在于提供一种可显著提高胚胎神经干细胞的纯度、细胞活力、细胞增殖速度并可抑制胚胎神经干细胞的分化而维持其多能分化潜能的神经干细胞的方法。本发明通过提供一种有效提取胚胎神经干细胞的方法,可方便、高效地提取大量高纯度和高活力的胚胎神经干细胞;同时,通过提供一种培养基的配方和组成成分的比例、浓度,可配制体外培养所提取的胚胎神经干细胞所需的培养基,可有效促进体外培养的胚胎神经干细胞的增殖并维持其细胞活力和多能分化潜能。
为实现上述目的,本发明采用如下技术方案:
一种体外提取培养胚胎神经干细胞的方法,包括以下步骤:
取出生24h内的新生鼠,脱颈处死后,迅速浸入75v/v%乙醇消毒,0.9w/v%氯化钠溶液冲洗,剥离皮肤,暴露脊柱,用无菌剪刀于上颈椎和骶椎处剪断脊柱,取出脊柱干部;用宽嘴镊子将脊柱内的脊髓挤出,放入0.9w/v%氯化钠溶液中,显微镜下剥离脊膜,将脊髓剪成1mm3的组织块,过滤后放入离心管中;以体积比1:1的比例加入0.25w/v%胰蛋白酶边吹打边消化20分钟,加入DMEM/F12培养基10mL中止消化,细胞滤网过滤后离心,倒掉上清液,收集细胞,将细胞以3×109的密度种植于75cm2的细胞培养皿中,加入胚胎神经干细胞完全培养基3mL,在37℃、含5%CO2的细胞培养箱中进行培养。
所述胚胎神经干细胞完全培养基配方包括DMEM/F12、谷氨酰胺、赖氨酸、B-27添加物、N2添加物、青霉素、链霉素、bFGF、EGF、胰岛素、小分子抑制剂CHIR99021。
所述胚胎神经干细胞完全培养基的制备方法为:将500mL DMEM/F12培养基吸除20mL,向剩余的DMEM/F12培养基中加入B-27添加物10mL、N2添加物5mL、青霉素2.5mL、链霉素2.5mL;终浓度10mM的谷氨酰胺、5μmol/mL的赖氨酸、20ng/mL 的bFGF、20ng/mL 的EGF、5μg/mL的胰岛素、5mM的小分子抑制剂CHIR99021 ,共同制备成500mL的胚胎神经干细胞完全培养基。
本发明的优点在于:
1、可提高提取胚胎干细胞的纯度和效率;
2、可显著维持并促进胚胎神经干细胞的体外存活能力和增殖能力,维持其生命活力;
3、可有效维持胚胎神经干细胞的多能分化潜能,不会导致体外培养的胚胎神经干细胞发生分化;
4、可有效降低体外培养的胚胎神经干细胞被污染的发生概率。
5、培养基配制方法简单。
附图说明
图1为细胞培养3天的光学显微镜观察图(200×);
图2为细胞培养7天的光学显微镜观察图(200×);
图3为试验组和对照组细胞增殖率比较情况;
图4为试验组和对照组胚胎神经干细胞纯度比较情况;
图5为免疫荧光检测胚胎神经干细胞特异性表面抗原Nestin表达。
具体实施方式
实施例1 胚胎神经干细胞的提取及培养基制备
取出生24h内的新生鼠,脱颈处死后,迅速浸入75v/v%乙醇消毒,0.9w/v%氯化钠溶液冲洗,剥离皮肤,暴露脊柱,用无菌剪刀于上颈椎和骶椎处剪断脊柱,取出脊柱干部;采用“挤牙膏”的方式用宽嘴镊子将脊柱内的脊髓挤出,放入0.9w/v%氯化钠溶液中,显微镜下剥离脊膜,将脊髓剪成1mm3的组织块,过滤后放入离心管中;按照体积比1:1的比例加入0.25w/v%胰蛋白酶边吹打边消化20分钟,加入DMEM/F12培养基10mL中止消化,细胞滤网过滤后离心,倒掉上清液,收集细胞。配制胚胎干细胞完全培养基,配方包括DMEM/F12、谷氨酰胺、赖氨酸、B-27添加物、N2添加物、青霉素、链霉素、bFGF、EGF、胰岛素、小分子抑制剂CHIR99021。制备方法:将500mL DMEM/F12培养基吸除20mL,向剩余的DMEM/F12培养基中加入B-27添加物10mL、N2添加物5mL、青霉素2.5mL、链霉素2.5mL、谷氨酰胺10mM、赖氨酸5μmol/mL、bFGF 20ng/mL、EGF 20ng/mL、胰岛素5μg/mL、小分子抑制剂CHIR99021 5mM(以上为终浓度),共同制备成500mL的胚胎神经干细胞完全培养基。
实施例2 细胞培养
将上述配制的培养基加入收集的细胞中进行培养。将细胞以3×109的密度种植于75cm2的细胞培养皿中,加入胚胎神经干细胞完全培养基3mL,在37℃、含5%CO2的细胞培养箱中进行培养。培养3天后可见大量胚胎神经干细胞,部分细胞聚集成球,增殖迅速(图1),培养7天后可见大量胚胎神经干细胞聚集成悬浮球,增殖迅速,细胞形态未发生改变,符合胚胎神经干细胞的生物学特性,未分化(图2)。
采用CCK8细胞增殖试剂盒检测细胞培养3天、6天、9天和12天后的细胞增殖率,发现采用本发明所提供技术培养的胚胎神经干细胞的增殖率在培养后6天开始显著高于现有技术(对照组)采用取大脑海马区的胚胎干细胞并使用商品化的培养基(赛叶生物公司,货号MUAES-90011)所培养的胚胎神经干细胞(图3)。
采用流式细胞仪对所培养的胚胎神经干细胞在培养后3天、6天、9天和12天进行细胞分选,计算胚胎神经干细胞纯度,发现本发明所提供技术培养的胚胎神经干细胞的纯度显著高于现有技术(对照组)所培养的胚胎神经干细胞(图4)。
对胚胎神经干细胞表面特异性抗原Nestin采用免疫荧光技术检测,发现采用本发明体外培养的胚胎神经干细胞所形成的神经球Nestin表达呈显著阳性(绿色),说明本发明体外培养的胚胎神经干细胞未发生分化(图5)。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (1)
1.一种体外提取培养胚胎神经干细胞的方法,其特征在于,包括以下步骤:
取出生24h内的新生鼠,脱颈处死后,迅速浸入75v/v%乙醇消毒,0.9w/v%氯化钠溶液冲洗,剥离皮肤,暴露脊柱,用无菌剪刀于上颈椎和骶椎处剪断脊柱,取出脊柱干部;用宽嘴镊子将脊柱内的脊髓挤出,放入0.9w/v%氯化钠溶液中,显微镜下剥离脊膜,将脊髓剪成1mm3的组织块,过滤后放入离心管中;以体积比1:1的比例加入0.25w/v%胰蛋白酶边吹打边消化20分钟,加入DMEM/F12培养基10mL中止消化,细胞滤网过滤后离心,倒掉上清液,收集细胞,将细胞以3×109的密度种植于75cm2的细胞培养皿中,加入胚胎神经干细胞完全培养基3mL,在37℃、含5%CO2的细胞培养箱中进行培养;
所述胚胎神经干细胞完全培养基配方包括DMEM/F12、谷氨酰胺、赖氨酸、B-27添加物、N2添加物、青霉素、链霉素、bFGF、EGF、胰岛素、小分子抑制剂CHIR99021;
所述胚胎神经干细胞完全培养基的制备方法为:将500mL DMEM/F12培养基吸除20mL,向剩余的DMEM/F12培养基中加入B-27添加物10mL、N2添加物5mL、青霉素2.5mL、链霉素2.5mL;终浓度10mM的谷氨酰胺、5μmol/mL的赖氨酸、20ng/mL 的bFGF、20ng/mL 的EGF、5μg/mL的胰岛素、5mM的小分子抑制剂CHIR99021 ,共同制备成500mL的胚胎神经干细胞完全培养基。
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