CN109280634B - 一种内耳毛细胞体外分离培养的方法 - Google Patents
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Abstract
本发明公开了一种内耳毛细胞体外分离培养的方法,所述方法为:将耳蜗基底膜毛细胞面朝上放置在提前用层粘连蛋白包被的四孔细胞培养板上,然后加内耳毛细胞培养基,每间隔24小时更换培养液,获得内耳毛细胞;所述内耳毛细胞培养基为含体积终浓度2‑15%马血清、体积终浓度2‑15%胎牛血清、体积终浓度1‑2%N‑2细胞因子添加剂的高糖DMEM培养基。本发明方法可以更加高效的分离得到完整的耳蜗基底膜;利用本发明的培养条件,基底膜毛细胞的形态更接近在体状态,外毛以及内毛排列更完整;同时相比传统方法(10%胎牛血清的高糖DMEM培养基),本发明方法可以延长基底膜毛细胞一倍以上的生存时间。
Description
(一)技术领域
本发明涉及一种内耳感觉毛细胞的分离及体外培养方法。
(二)背景技术
毛细胞作为内耳感觉细胞,可以将声音的振动信号转化为电信号。毛细胞的损伤以及缺失就会造成聋病的发生。造成毛细胞损伤原因有许多,包括药物,环境,遗传因素以及年龄等。内耳毛细胞分布在哺乳动物的内耳耳蜗、椭圆囊、壶腹脊、球囊,位于椭圆囊的毛细胞主要感受线速度,而位于壶腹脊的毛细胞则感受旋转角速度,只有位于耳蜗的毛细胞才能将传入内耳的声音振动信号转化为电信号,最终由耳蜗的传入神经传递到大脑。耳蜗包埋在坚硬的颞骨中,加上耳蜗结构精细复杂,使得毛细胞解剖分离十分困难,造成了毛细胞体外研究面临很大的局限。
内耳中的毛细胞受损后在鱼类等可以再生,但在哺乳动物中一旦受损无法再生。毛细胞的体外培养技术可以为研究毛细胞分化发育机制提供基础。然而,目前毛细胞体外的分离培养有待进一步完善,目前的技术无法长时间体外培养毛细胞,维持其较高的存活率(参考Ding D等:Cisplatin ototoxicity in rat cochlear organotypiccultures.Hearing research 2011,282(1-2):196-203.Ou HC等:"In-bone"utriclecultures--a simplified,atraumatic technique for in situ cultures of the adultmouse(Mus musculus)utricle.Otology&neurotology:official publication of theAmerican Otological Society,American Neurotology Society&European Academy ofOtology and Neurotology 2013,34(2):353-359.)。现阶段各个实验室比较常用的体外培养体系包括了添加血清和不添加血清的两种,不添加血清的培养方法采用MEM培养基(sigma)添加10%BSA(sigma),而血清培养体系则采用DMEM(sigma)以及10%胎牛血清(sigma);利用这两种传统的培养方法离体培养的内耳毛细胞形态维持时间仅为3-5天,且随着培养时间的延长毛细胞数量逐渐降低、毛细胞纤毛束的形态逐渐消失。
(三)发明内容
本发明目的是提供一种优化的内耳毛细胞体外分离培养方法,该方法和传统分离培养方法相比,可以显著延长哺乳动物内耳毛细胞体外培养存活时间,有助于体外毛细胞研究工作的开展。
本发明采用的技术方案是:
本发明提供一种内耳毛细胞体外分离培养的方法,所述方法为:将小鼠麻醉处死后分离耳蜗,解剖镜下小心剥出耳蜗基底膜;耳蜗基底膜毛细胞面朝上放置在提前用层粘连蛋白包被的四孔细胞培养板上,然后每孔小心添加(优选350μl)37℃预热的内耳毛细胞培养基;将培养板转移至细胞培养箱(37℃、5%CO2)中进行培养,每间隔24小时更换培养液,获得结构清晰、排列整齐的内耳毛细胞;所述内耳毛细胞培养基为含体积终浓度2-15%马血清、体积终浓度2-15%胎牛血清、体积终浓度1-2%N-2细胞因子添加剂的高糖DMEM培养基。
进一步,优选所述内耳毛细胞培养基为含体积终浓度5%马血清、体积终浓度5%胎牛血清、体积终浓度1%N-2细胞因子添加剂的高糖DMEM培养基。
进一步,所述层粘连蛋白包被细胞培养板的方法为:将层粘连蛋白,多聚鸟氨酸和胎牛血清按照体积比2:2:1均匀混合,然后将混合液小心加入培养板中;随后用封口膜封闭培养板的边缘,放置到37℃培养箱中孵育30min,完成包被。
进一步,培养液更换方法为:每次用新鲜内耳毛细胞培养基更换一半体积的培养液。
所述高糖DMEM培养基为HyClone SH30243.01。
选择3-5天的新生小鼠作为实验的材料,这个年龄段的小鼠内耳尚未钙化,适合解剖。
与现有技术相比,本发明的有益效果主要体现在:本发明方法可以更加高效的分离得到完整的耳蜗基底膜;利用本发明的培养条件,基底膜毛细胞的形态更接近在体状态,外毛以及内毛排列更完整;同时相比传统方法(10%胎牛血清的高糖DMEM培养基),本发明方法可以延长基底膜毛细胞一倍以上的生存时间。相比利用常规毛细胞培养基进行毛细胞的体外培养方法(只能维持毛细胞形态3-5天),本发明方法培养过程中基底膜边缘上皮细胞生长抑制明显,可以维持内耳毛细胞较好形态至少一周,最长时间可达10天,体外培养时间显著延长。
(四)附图说明
图1小鼠内耳解剖结构图,A为尚未完全钙化的新生C57/BL6小鼠内耳;B为已经钙化的成年C57/BL6小鼠内耳。
图2基底膜解剖示意图。
图3不同培养基体外培养3天的小鼠耳蜗基底膜倒置显微镜图。A为内耳毛细胞培养基;B为对照3常规培养基,C为对照2常规培养基,D为对照1常规培养基。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明实施例百分比除特别说明外,均为体积百分浓度。
实施例1小鼠基底膜毛细胞的分离以及体外培养
1、利用出生后3-5天的C57BL/6J小鼠作为实验材料。
2、在解剖耳蜗之前要提前用层粘连蛋白包被细胞培养四孔板,在超净台里将层粘连蛋白(Sigma-Aldrich 114956-81-9),多聚鸟氨酸(Sigma-Aldrich 27378-49-0)和胎牛血清(Gibco 16000044)按照体积比2:2:1均匀混合,然后添加300μl混合液到四孔板每个孔中,封口膜封闭四孔板的边缘后,放到37℃的培养箱中孵育30min;随后移去包被液,用4℃预冷的PBS冲洗3遍备用;向四孔板的每个孔中添加250μl内耳毛细胞培养基(配方为:高糖DMEM培养基(HyClone SH30243.01)+5%马血清(ThermoFisher 16050130)+5%胎牛血清(Gibco 16000044)+1%N-2细胞因子添加剂(ThermoFisher 16000044)),结果见图3中A。
对照1:在层粘连蛋白包被的四孔板其他孔中添加常规培养基(高糖DMEM培养基(HyClone SH30243.01)+10%胎牛血清(Gibco 16000044)),结果见图3中D。
对照2:在层粘连蛋白包被的四孔板其他孔中添加常规培养基(高糖DMEM培养基(HyClone SH30243.01)+5%马血清(ThermoFisher 16050130)+5%胎牛血清(Gibco16000044)),结果见图3中C。
对照3:在层粘连蛋白包被的四孔板其他孔中添加常规培养基(高糖DMEM培养基(HyClone SH30243.01)+5%马血清(ThermoFisher 16050130)+5%胎牛血清(Gibco16000044)+1%N-1细胞因子添加剂(Sigma N6530),结果见图3中B。
3、将小鼠安乐处死后,在70%酒精浸泡1min,然后转移到4℃预冷的含1×双抗(BI03-031-1B)的无菌磷酸缓冲液(PBS,常规配制,pH值7.4)中,打开颞骨分离内耳(如图1中A所示),在解剖镜下进行用#5精细镊子将附着的其他组织去除干净(如图2)。
4、随后将内耳转移到新的4℃预冷PBS中,解剖镜下剥离壶腹脊、椭圆囊、球囊等,之后去除耳蜗蜗轴的螺旋神经节,最后用#5镊子移去基底膜上血管纹以及盖膜等。注意解剖时一定要保证耳蜗基底膜不发生蜷曲,解剖时间尽量控制在10min之内。
5、解剖后,将耳蜗基底膜轻轻移到步骤2准备好的四孔板里,保证基底膜毛细胞面朝上;待基底膜铺展开后,分别每孔添加350μl 37℃预热的内耳毛细胞培养基或常规培养基。添加时先用移液枪轻轻从四孔板的边缘轻轻添加250μl,之后的100μl则轻轻的滴加到基底膜上;最后将孔板放入37℃、5%CO2培养箱中进行培养。
6、24h后首次更换培养液,换液方式为半量换液,即每次换掉一半旧的培养基之后再添加一半新的培养基。培养的过程中在倒置显微镜下进行镜检观察耳蜗毛细胞的排列情况。如图3(A,B,C,D)所示,图3中A表明利用本发明方法体外培养毛细胞72小时后结构完整、细胞形态清晰。图3中B表明将培养基中的1%N-2细胞因子添加剂替换成相同浓度的1%N-1细胞因子添加剂,其他培养基成分不变。相对普通的培养基,毛细胞的缺失较少,但是比A中缺失明显。图3中C没有添加1%N-2细胞因子添加剂的培养基,相对普通培养基,毛细胞缺失较少,但是比A中缺失更明显。图3中D普通培养基培养的毛细胞已经出现严重缺失。相比利用常规毛细胞培养基进行毛细胞的体外培养方法(只能维持毛细胞形态3-5天),本发明方法培养过程中基底膜边缘上皮细胞生长抑制明显,可以维持内耳毛细胞较好形态至少一周,最长时间可达10天,体外培养时间显著延长。
Claims (4)
1.一种内耳毛细胞体外分离培养的方法,其特征在于所述方法为:将小鼠耳蜗基底膜的毛细胞面朝上,放置在层粘连蛋白包被的细胞培养孔板上,添加37℃预热的内耳毛细胞培养基,在37℃、5%CO2条件下进行培养,间隔24h更换新鲜培养液,获得内耳毛细胞;所述内耳毛细胞培养基为含体积终浓度2-15%马血清、体积终浓度2-15%胎牛血清、体积终浓度1-2%N-2细胞因子添加剂的高糖DMEM培养基。
2.如权利要求1所述内耳毛细胞体外分离培养的方法,其特征在于所述内耳毛细胞培养基为含体积终浓度5%马血清、体积终浓度5%胎牛血清、体积终浓度1%N-2细胞因子添加剂的高糖DMEM培养基。
3.如权利要求1所述内耳毛细胞体外分离培养的方法,其特征在于所述层粘连蛋白包被细胞培养孔板的方法为:将层粘连蛋白,多聚鸟氨酸和胎牛血清按照体积比2:2:1均匀混合,然后将混合液小心加入细胞培养孔板中;随后用封口膜封闭培养孔板的边缘,于37℃的培养箱中孵育30min,完成包被。
4.如权利要求1所述内耳毛细胞体外分离培养的方法,其特征在于培养液更换方法为:每次用新鲜内耳毛细胞培养基更换一半体积的培养液。
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