CN104164365A - 一种体外细胞接触式共培养装置及其培养操作方法 - Google Patents
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Abstract
本发明公开了一种体外细胞接触式共培养装置及其培养操作方法,其是在培养盒体的底端设有盒底,盒体的上端设有盒盖,培养盒体内设有细胞培养槽,所述的培养盒体设为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底直径相同,培养槽的中部设有隔离膜,隔离膜固定于培养槽的中部将培养槽分隔为上下两个培养槽,本发明巧妙的运用了聚酸酯膜将培养槽分隔成上下培养槽,并在其上下的两侧分别接种,翻转后再置于细胞培养盒中,实现了细胞实质性的接触式共培养,便于研究一种细胞代谢物对另一种细胞的影响以及直接接触而导致的胞间作用。
Description
技术领域
本发明涉及一种细胞培养装置,确切的说是一种体外细胞接触式共培养装置及其培养操作方法。
背景技术
在动物体内,细胞处于一个高度信息化的微环境中,其间存在各种复杂的物理化学信号,细胞对各种信号刺激做出响应,以实现其特定的生物学功能。细胞与这种微环境间的相互作用包括来自周围细胞、细胞外基质和可溶性因子的各种复杂的生物化学、生物力学和生物电学信号。其中,细胞与细胞之间是通过直接接触或旁分泌可溶性因子而发生相互作用。20世纪80年代后期,为了建立更类似于体内环境的培养体系,尽可能使体外环境与体内环境相吻合,从而使细胞间能相互沟通信息,相互支撑生长增殖,人们在细胞培养技术的基础上发展出了细胞共培养技术。细胞共培养技术是将2种或2种以上的细胞共同培养于同一环境中,由于其具有更好地反映体内环境的优点,所以这种方法被广泛应用于现代细胞研究中。
目前,现有的细胞共培养体系主要通过两种方法建立:
一是直接共培养体系,是将两种或两种以上的细胞按照一定比例同时或分别接种于细胞培养皿或板孔中,在同一培养环境中进行共同培养,不同种类的细胞之间直接接触。常用的是细胞培养皿和不同孔径的细胞培养板,或者把细胞培养皿或板孔分割成不同空间,把不同细胞接种在底部。
二是间接共培养体系,是将两种或两种以上的细胞分别接种于不同的载体上,然后将这两种载体置于同一培养环境之中,使不同种类的细胞共用同一种培养体系而不直接接触,从而达到研究细胞的分泌或代谢产物对目的细胞生长、迁移、增殖、分化等生物学行为影响的目的。
然而,现有的细胞共培养装置不能满足不同形式和不同目的的培养。传统接触式共培养有的是不同细胞混养在一起,很难调控体系中同型与异型细胞间相互作用比例,且后期深入研究时也很难有效的把不同种细胞分离开,所以不好检测两类细胞增殖分化以及相关基因的表达。有的细胞是生长在分割出不同空间的同一细胞培养皿或板孔中,由于细胞都是贴底壁生长,这种情况下只能实现不同细胞间分泌物质的交流,但并不能实现不同细胞间的实质性接触。就象非接触式共培养体系仅能部分模拟体内细胞间的相互作用,缺乏由异种细胞间直接接触而导致的胞间作用。所以,在细胞体外培养过程中,如何有效模拟体内微环境,调控不同细胞之间、细胞与细胞外微环境间的相互作用,对于细胞形态与功能的维持以及细胞间的相互影响至关重要。
发明内容
本发明目的就是克服上述缺陷,提供一种细胞交流更加充分,可以实现细胞实质性接触式共培养,细胞换液更加方便的体外细胞接触式共培养装置。
本发明的另一个目的就是提供该培养装置的使用操作方法。
本发明的方案是由培养盒体、盒底及盒盖组成,培养盒体的底端设有盒底,盒体的上端设有盒盖,培养盒体内设有细胞培养槽,所述的培养盒体设为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底直径相同,培养槽的中部设有隔离膜,隔离膜为一具有通透性能的聚碳酸酯膜,固定于培养槽的中部将培养槽分隔为上下两个培养槽。
所述的隔离膜为聚碳酸酯膜,其孔径为3-8μm,能够使细胞的分泌物及各种营养因子及其他分子物质通过,而阻止细胞穿过。
本发明的有益效果:首先一是巧妙的运用了聚酸酯膜将培养槽分隔成上下培养槽,并在其上下的两侧分别接种,翻转后再置于细胞培养盒中,实现了细胞实质性的接触式共培养,便于研究一种细胞代谢物对另一种细胞的影响以及直接接触而导致的胞间作用;二是容易控制体系中不同细胞接种比例和细胞间相互作用比例,且后期深入研究时也很容易有效地把不同种细胞分离开,易于检测两类细胞增殖分化以及相关基因的表达;三是两种细胞生长在细胞培养槽的聚碳酸酯膜两侧,取拿细胞培养槽方便易于细胞的换液工作,不易污染。
下面结合附图作进一步详细说明。
附图说明
图1为培养装置结构示意图;
图2为细胞培养槽结构示意图;
图3为细胞培养盒外形示意图;
图4为细胞共培养装置使用状态参考图。
具体实施方式
图1-4中示出的培养盒体2的底端设有盒底1,盒体的上端设有盒盖3,培养盒体内设有细胞培养槽5,所述的培养盒体为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底相同,细胞培养槽的中部设有隔离膜4,隔离膜为一具有通透性能的聚碳酸酯膜,隔离膜固定于培养槽的中部,将培养槽分隔为上下两个培养槽。
图4中,先将C2C12成肌细胞悬液6接种在隔离膜的上侧,盖上盒盖,放置在培养箱中孵育,待24-48小时后,细胞贴壁增殖成单层细胞后,贴附在隔离膜上壁上,将细胞培养槽反转过来,使贴壁的C2C12细胞翻转在隔离膜的下侧面,再将3T3-L成纤维细胞7接种在隔离膜的上侧面。
共培养装置的操作方法步骤如下
1、向C2C12细胞培养瓶中加入1ml浓度为0.25%胰蛋白酶液消化细胞,再加入浓度为10%胎牛血清的高糖DMEM液体培养基5ml,用胶头滴管搅拌混合均匀,然后将混合液移入10ml离心管中,在离心机上离心5min,弃上清液,离心机转速为1500转/min,使其配制成浓度为3×104个/ml细胞悬液;
2、将配制好的细胞悬液置入细胞培养槽内的隔离膜的上侧,在隔离膜上侧的细胞悬液中接种C2C12成肌细胞悬液,将细胞培养槽置于细胞培养盒中,盖上细胞培养盒盖,放在超净工作台上缓慢摇晃以混匀细胞,然后将细胞培养盒放在37℃的恒温培养箱中孵育,待24-48小时后,细胞贴壁增殖成单层细胞后,粘附在隔离膜上壁上;
3、将余下的培养液用吸液枪吸出打到废液缸后,把细胞培养槽反转过来,倒置于细胞培养盒内,使贴壁的C2C12细胞翻转在隔离膜的下侧面;
4、按照上述同样的方法将1.5g碳酸氢钠、4.5g葡萄糖混合离心后,用浓度10%胎牛血清的高糖DMEM液体培养基5ml,将其配制成浓度为4×105个/ml细胞悬液;
5、将配制好的细胞悬液置入细胞培养槽隔离膜的上侧,在隔离膜上侧的细胞悬液中接种3T3-L成纤维细胞,这样就形成了在细胞培养槽的聚碳酸脂膜下侧接种C2C12细胞,上侧接种3T3-L细胞的细胞直接接触式共培养状态;
6、将此装置放入37℃的恒温培养箱内孵育,每隔一天为两种细胞换液1次。
由于生长在聚碳酸酯膜的两侧,两种细胞能直接接触,细胞产物也可以充分交流,可以在培养的不同阶段,观察两种细胞的生长情况,并分析相互影响的结果。
本装置实验时可用于两种细胞的接触式共培养,作为载体的通透性膜有利于相邻细胞间的直接交流,很好的模拟了体内细胞共存的情况。
所述的C2C12细胞为一种商业化小鼠成肌细胞系,3T3-L细胞是一种商业化细胞,是从小鼠胚胎克隆的特种细胞系,市场均有售,也可从中国科学院上海细胞生物学研究所;高糖DMEM液体培养基:市场有售,货号:12491-015,或购自Gibco公司;所述的超净工作台,市场有售或购于苏州净化有限责任公司。
Claims (3)
1.一种体外细胞接触式共培养装置,由培养盒体、盒底及盒盖组成,其特征在于:所述培养盒体(2)的底端设有盒底(1),盒体的上端设有盒盖(3),培养盒体内设有细胞培养槽(5)。
2.根据权利要求1所述的体外细胞接触式共培养装置,其特征在于:所述的培养盒体为圆锥陀螺形,其上端的直径大于底端的直径,细胞培养槽为圆桶形,圆桶的上下直径与培养盒体的盒底相同,细胞培养槽的中部设有隔离膜(4),隔离膜为一具有通透性能的聚碳酸酯膜,隔离膜固定于培养槽的中部,将培养槽分隔为上下两个培养槽。
3.根据权利要求1或2所述的共培养装置的培养操作方法,其特征在于:它包括下列步骤:
(1)向C2C12细胞培养瓶中加入1ml浓度为0.25%胰蛋白酶液消化细胞,再加入浓度为10%胎牛血清的高糖DMEM液体培养基5ml,用胶头滴管搅拌混合均匀,然后将混合液移入10ml离心管中,在离心机上离心5min,弃上清液,离心机转速为1500转/min,使其配制成浓度为3×104个/ml细胞悬液;
(2)将配制好的细胞悬液置入细胞培养槽内的隔离膜的上侧,在隔离膜上侧的细胞悬液中接种C2C12成肌细胞悬液,将细胞培养槽置于细胞培养盒中,盖上细胞培养盒盖,放在超净工作台上缓慢摇晃以混匀细胞,然后将细胞培养盒放在37℃的恒温培养箱中孵育,待24-48小时后,细胞贴壁增殖成单层细胞后,粘附在隔离膜上壁上;
(3)将余下的培养液用吸液枪吸出打到废液缸后,把细胞培养槽反转过来,倒置于细胞培养盒内,使贴壁的C2C12细胞翻转在隔离膜的下侧面;
(4)按照上述同样的方法将1.5g碳酸氢钠、4.5g葡萄糖混合离心后,用浓度10%胎牛血清的高糖DMEM液体培养基5ml,将其配制成浓度为4×105个/ml细胞悬液;
(5)将配制好的细胞悬液置入细胞培养槽隔离膜的上侧,在隔离膜上侧的细胞悬液中接种3T3-L成纤维细胞,这样就形成了在细胞培养槽的聚碳酸脂膜下侧接种C2C12细胞,上侧接种3T3-L细胞的细胞直接接触式共培养状态;
(6)将此装置放入37℃的恒温培养箱内孵育,每隔一天为两种细胞换液1次。
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