CN106963987B - 一种经由细胞片层获得的导电细胞外基质复合薄膜及其制备方法 - Google Patents
一种经由细胞片层获得的导电细胞外基质复合薄膜及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种经由细胞片层获得的导电细胞外基质复合薄膜及其制备方法,该方法如下:主要是在可光致细胞脱附的细胞培养表面(如TiO2纳米点薄膜)上进行培养细胞,在培养的过程中加入纳米导电物质,如石墨烯纳米片、石墨烯纳米点等,这些纳米导电颗粒会被细胞内吞,导电物质分散在细胞内及细胞间,当细胞片层融合后,通过光致细胞脱附,获得一层完整的细胞片层,将其清洗后再进行脱细胞进而获得导电细胞外基质复合薄膜。本发明方法制得的导电细胞外基质复合薄膜,可应用于组织工程等领域。此外本发明的制备方法,工艺简单,易于实现,有利于进行推广应用。
Description
技术领域
本发明涉及组织工程领域,具体涉及一种经由细胞片层获得导电细胞外基质复合薄膜及其制备方法。
背景技术
细胞外基质是组织的基本组成部分之一,由一些糖蛋白、结构蛋白及粘着蛋白组成,可对细胞的基本生命活动进行全方位的生物学调控,如对细胞的形状、生长、迁移和分化,胚胎的发育以及受损组织或器官的修复等有至关重要的作用。有研究报道胶原蛋白及透明质酸等模拟的细胞外基质环境和电刺激的协同作用能有效的促进人骨髓间充质干细胞向成骨方向分化[Hess R,Jaeschke A,Neubert H,et al.Synergistic effect ofdefined artificial extracellular matrices and pulsed electric fields onosteogenic differentiation of human MSCs.Biomaterials,2012,33(35):8975-8985]。虽然天然的生物材料(胶原及透明质酸等)具有一定的生物相容性,但是来源有限,价格相对昂贵,同时具有生物排异性。而支架自身的导电性是决定可否进行电刺激的关键。因此,如何获得细胞分泌的细胞外基质与导电物质的复合材料在组织工程具有很强的实际应用意义和研究价值。
细胞在体外培养过程中就可在与基板连接处分泌出一层细胞外基质。如果进行胰酶处理,在细胞脱离培养表面之前细胞外基质就会被消化掉,无法获得所需的细胞外基质层。而单纯的机械剥离,则会使细胞外基质超微结构发生一定改变,影响其后续的功能性。
本发明在近年来报道的光致细胞薄层获取技术基础上,开发了一种经由细胞片层获得导电细胞外基质复合薄膜的方法。该方法利用光致细胞薄层脱附技术能够获得具有细胞外基质含量高、活性功能良好的细胞薄层的特点[Y.Hong,M.F.Yu,W.J.Weng,K.Cheng,H.M.Wang,J.Lin.Light-induced cell detachment for cell sheettechnology.Biomaterials,2013,34(1):11-18],在细胞培养过程中加入不同的导电物质,并经过一系列的处理获取了一种导电细胞外基质复合薄膜。该方法获得的细胞外基质薄膜保持了原有的超微结构和组成成分,同时具有良好的机械性能,并且导电物质均匀分布于复合薄膜中,可应用于组织工程等领域。本发明的制备方法,工艺简单,易于实现,有利于进行推广应用,所得到的复合薄膜具有良好的导电性、生物相容性及组织修复特性。
发明内容
本发明的目的在于提供一种经由细胞片层获得导电细胞外基质复合薄膜及其制备方法,所述的导电细胞外基质复合薄膜可通过控制纳米导电物质的种类、浓度及添加时间去调控细胞外基质的结构及导电性能。
一种经由细胞片层获得导电细胞外基质复合薄膜的方法,包括如下步骤:
(a).对可见光致细胞脱附的细胞培养表面以紫外光照或蒸气消毒方式消毒;
(b).将上述经处理后的表面作为体外细胞培养表面,将事先培养在培养瓶中的细胞进行脱壁处理,以800~1300r/min离心2~6min后,用α-MEM培养基混悬,并用血细胞计数仪计数,在细胞培养表面以5×104~2×106个/cm2的密度种植细胞,加入细胞培养基,并放入37℃恒温及5%二氧化碳的细胞培养箱中培养7~15天;1~3天换液一次,在细胞脱附前1~5天加入1~100μg/mL纳米导电物质,纳米导电物质会被细胞内吞进去,最终在细胞培养表面上形成完整的含有导电物质的细胞片层;
(c).将经上述培养后的培养表面移入PBS中,通过波长于365nm的紫外光光照5~30min,使培养后的细胞或细胞薄层脱附,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗;
(d).将细胞片层浸泡于去离子水中,置于-80℃冷冻30~60min,取出后在25~37℃解冻20~45min,冷冻-解冻循环重复3~10次,之后用去离子水清洗,获得导电细胞外基质复合薄膜。
上述技术方案中,所述的细胞培养表面采用TiO2纳米点薄膜。
所述的纳米导电物质为石墨烯纳米片、石墨烯纳米点、纳米碳纤维、单壁碳纳米管、多壁碳纳米管、纳米碳颗粒、碳量子点、纳米金颗粒、纳米银颗粒中的一种或多种。其中,所述的石墨烯纳米片尺寸为0.5~2μm,厚度为0.8~2nm;石墨烯纳米点的尺寸为5~10nm;纳米碳纤维的直径为50~200nm;单壁碳纳米管的直径为1~2nm,长度1~3μm;多壁碳纳米管的直径为1~50nm,长度0.5~2μm;碳量子点的尺寸为3~5nm;纳米金颗粒的直径为10~50nm;纳米银颗粒的直径为10~100nm。
经上述方法制备获得的一种导电细胞外基质复合薄膜,为典型的均匀且致密的纤维网状结构,厚度为3~10μm。
本发明的薄膜及制备方法具有如下特点:
1)采用表面含有TiO2纳米点薄膜的基板进行细胞培养,通过紫外光灯照射,使含有导电物质的细胞片层从基板上脱附下来,减少细胞片层的机械损伤,从而获得完整的含有导电物质的细胞片层。
2)纳米导电物质不仅存在于细胞内而且存在于细胞连接处,最终获取的是导电物质均匀分布的导电复合薄膜。
3)所获取的导电细胞外基质复合薄膜不仅保存了细胞外基质的重要组成成分,而且具有良好的导电特性。
本发明所涉及的导电细胞外基质复合薄膜的制备过程,无论是细胞的体外培养,纳米导电物质与细胞的复合,还是细胞片层的脱附,都是比较简洁易行的,对设备没有过高的要求。本发明的导电细胞外基质复合薄膜,保持了细胞外基质的拓扑结构,具有良好的生物相容性及导电性,为培养同种或异种细胞提供了有利的微环境,有利于细胞的粘附及增值,对细胞的分化也产生影响,导电纳米颗粒的存在有利于外界电刺激的施加,可有效调控对于电信号敏感的神经细胞行为及诱导干细胞的定向分化等,可应用于组织工程等领域。此外本发明的制备方法,工艺简单,易于实现,有利于进行推广应用。
附图说明
图1是导电细胞外基质复合薄膜的表面形貌图。
图2是导电细胞外基质复合薄膜的拉曼图。
图3是导电细胞外基质复合薄膜的电导率图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。
实施例1
(1)对涂有TiO2的纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以800r/min离心6min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以1×105个/cm2细胞密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养10天,2天后进行第一次换液,之后每2天换液一次,在细胞脱附前1天加入100μg/mL石墨烯纳米片,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照30min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗2遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻30min,取出后在37℃解冻20min。冷冻-解冻循环重复10次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
本例制得的细胞外基质薄膜的表面形貌图如图1所示,可以看出导电细胞外基质薄膜结构致密,图2显示石墨存在于复合薄膜中,图3显示复合薄膜的导电性良好。
实施例2
(1)对涂有TiO2的纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1300r/min离心2min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以2×106个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养7天,2天后进行第一次换液,之后每2天换液一次,在细胞脱附前3天加入1μg/mL石墨烯纳米点,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照5min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻45min,取出后在30℃解冻30min。冷冻-解冻循环重复3次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
本例制得的细胞外基质薄膜生物相容性较好。
实施例3
(1)对涂有TiO2的纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1000r/min离心4min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以5×104个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养15天,2天后进行第一次换液,之后每3天换液一次,在细胞脱附前2天加入10μg/mL纳米碳纤维,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照15min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻60min,取出后在25℃解冻45min。冷冻-解冻循环重复3次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
实施例4
(1)对涂有TiO2的纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1200r/min离心5min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以5×105个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养8天,2天后进行第一次换液,之后每2天换液一次,在细胞脱附前4天加入2μg/mL单壁碳纳米管,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照20min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗2遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻40min,取出后在37℃解冻25min。冷冻-解冻循环重复6次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
实施例5
(1)对涂有TiO2纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1000r/min离心4min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以2.5×105个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养11天,2天后进行第一次换液,之后每2天换液一次,在细胞脱附前2天加入10μg/mL多壁碳纳米管,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照10min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻50min,取出后在30℃解冻40min。冷冻-解冻循环重复8次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
实施例6
(1)对涂有TiO2纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1000r/min离心4min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以2.5×105个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养11天,2天后进行第一次换液,之后每2天换液一次,在细胞脱附前4天加入15μg/mL纳米碳颗粒,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照10min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻45min,取出后在37℃解冻20min。冷冻-解冻循环重复9次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
实施例7
(1)对涂有TiO2纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1200r/min离心5min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以7×105个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养9天,2天后进行第一次换液,之后每2天换液一次,在细胞脱附前5天加入3μg/mL碳纳米点,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照15min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻45min,取出后在37℃解冻20min。冷冻-解冻循环重复9次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
实施例8
(1)对涂有TiO2纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1000r/min离心4min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以7×104个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养13天,2天后进行第一次换液,之后每3天换液一次,在细胞脱附前1天加入45μg/mL纳米金颗粒,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照10min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻45min,取出后在37℃解冻20min。冷冻-解冻循环重复9次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
实施例9
(1)对涂有TiO2纳米点薄膜的表面进行灭菌处理;
(2)将事先培养在培养瓶中的细胞进行脱壁处理,以1000r/min离心4min后,用α-MEM培养基混悬,并用血细胞计数仪计数,以1×105个/cm2的密度将细胞接种于细胞培养表面上,置于二氧化碳培养箱内进行高密度培养10天,2天后进行第一次换液,之后每3天换液一次,在细胞脱附前1天加入80μg/mL纳米银颗粒,最终在细胞培养表面上形成完整的细胞片层;
(3)通过波长365nm的紫外光光照30min,即可使培养后的含有导电物质的细胞片层脱附,获得一层完整的细胞片层,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗3遍。
(4)将细胞片层浸泡去离子水中,置于-80℃冷冻45min,取出后在37℃解冻20min。冷冻-解冻循环重复7次。之后用PBS缓冲液冲洗3遍,最后用去离子水清洗3遍,最终获得一层导电细胞外基质复合薄膜。
Claims (4)
1.一种经由细胞片层获得导电细胞外基质复合薄膜的方法,其特征在于,制备方法包括如下步骤:
(a). 对涂有TiO2纳米点薄膜的表面以紫外光照或蒸气消毒方式消毒;
(b). 将上述经处理后的表面作为体外细胞培养表面,在其表面以5×104~2×106个/cm2的密度种植细胞,加入细胞培养基,并放入37℃恒温及5%二氧化碳的细胞培养箱中培养7~15天;1~3天换液一次,在细胞脱附前1~5天加入1~100 μg/mL纳米导电物质,最终在细胞培养表面上形成完整的细胞片层;
(c). 将经上述培养后的培养表面移入PBS中,通过波长为365 nm的紫外光光照5~30min,使培养后的细胞或细胞薄层脱附,用PBS缓冲液以及去离子水对脱附后的细胞片层反复清洗;
(d). 将细胞片层浸泡于去离子水中,置于-80℃冷冻30~60 min,取出后在25~37℃解冻20~45 min,冷冻-解冻循环重复3~10次,之后用去离子水清洗,获得导电细胞外基质复合薄膜。
2.根据权利要求1所述的经由细胞片层获得导电细胞外基质复合薄膜的方法,其特征在于,所述的纳米导电物质为石墨烯纳米片、石墨烯纳米点、纳米碳纤维、单壁碳纳米管、多壁碳纳米管、纳米碳颗粒、碳量子点、纳米金颗粒、纳米银颗粒中的一种或多种。
3. 根据权利要求2所述的经由细胞片层获得导电细胞外基质复合薄膜的方法,其特征在于,所述的石墨烯纳米片尺寸为0.5~2 μm,厚度为0.8~2 nm;石墨烯纳米点的尺寸为5~10nm;纳米碳纤维的直径为50~200 nm;单壁碳纳米管的直径为1~2 nm,长度1~3μm;多壁碳纳米管的直径为1~50 nm, 长度0.5~2μm;碳量子点的尺寸为3~5 nm;纳米金颗粒的直径为10~50 nm;纳米银颗粒的直径为10~100 nm。
4.一种导电细胞外基质复合薄膜,其特征在于,采用如权利要求1所述的方法制备而成,厚度为3~10μm。
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