CN117106585A - 一种含细胞的微流控芯片及其制备方法和应用 - Google Patents
一种含细胞的微流控芯片及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于微流控仿生芯片技术以及材料科技与脑科技技术领域,具体涉及一种含细胞的微流控芯片及其制备方法和应用。所述芯片由小胶质细胞通道、神经元通道、骨骼肌细胞和外源炎症因子通道组成,本发明所设计的芯片,能够在体外模拟人神经炎症/帕金森病灶部位细胞间的相互作用关系,与传统的帕金森病动物模型相比,该模型将稳定的人源细胞株在微流控芯片中培养,使得芯片在便于重复构建、操控和长期连续观察的同时,能够实现对人帕金森病进行性发病过程的体外模拟,并满足对相关生物分子的检测和调控。
Description
技术领域
本发明属于微流控仿生芯片技术以及材料科技与脑科技技术领域,具体涉及一种含细胞的微流控芯片及其制备方法和应用。
背景技术
帕金森病是一种常见的神经系统退行性疾病,表现为静止性震颤、肌强直和运动迟缓等,在世界范围内,尤其是老年人中具有相当大的患者群体。帕金森病患者的根本器质性病理改变是中脑黑质区域多巴胺能神经元的进行性变性死亡,及由此导致的纹状体多巴胺含量减少,从而导致患者表现出一系列的震颤麻痹症状。迄今为止,帕金森病的致病机理尚未完全探明,其中已经阐明的机理之一是黑质细胞变性导致小胶质细胞非正常地暴露于神经黑色素中,使得小胶质细胞被激活而引发神经炎症,从而损伤多巴胺能神经元,导致多巴胺合成的受阻,宏观上引起肌肉强直,震颤麻痹等症状。
疾病模型是帕金森病研究的重要工具和手段,虽然目前已有以大鼠为实验动物通过注射方法构建多巴胺能神经元急性死亡的帕金森病模型构建方法,但是这些动物模型无法展示多巴胺的神经元的进行性和选择性损失,且也不会产生帕金森病患者特征性的路易体蛋白质块。
因此,在体外构建人源细胞的帕金森疾病模型对更精准深入地研究帕金森病及开发相关药物具有重要意义。
发明内容
有鉴于此,本发明的第一个目的是针对现有技术中存在的问题,提供一种便于重复构建和操控的体外帕金森疾病模型。
为了实现上述目的,本发明采用如下技术方案:
一种含细胞的微流控芯片,依次包括三个并列的细胞培养腔室:小胶质细胞腔室(1)、神经元腔室(2)和骨骼肌细胞腔室(3),且所述神经元腔室(2)位于小胶质细胞腔室(1)和骨骼肌细胞腔室(3)之间;每个细胞培养腔室两侧分别设置培养基入口通道(4)和废液出口通道(5);小胶质细胞腔室(1)与外源炎症因子通道(6)相联通;相邻细胞培养腔室间由微柱阵列(7)相隔。
微流控技术是一种对于微量流体精确操控的技术,可用于构建体外细胞培养的仿生芯片,其可以通过膜,壁等结构分割腔室,实现模拟体内真实结构的细胞分层培养。微流控平台能够满足分子生物学检测、显微观察、高通量药物筛选等要求,也便于重复构建和操控。
进一步的,所述小胶质细胞腔室(1)培养的细胞为人永生化小胶质细胞株HMC3(Human Microglia Clone 3);神经元腔室(2)培养的细胞为经人骨髓干细胞衍生的MNs分化得到的人运动神经元、经人多巴胺能神经元前体细胞LUHMES分化得到的人多巴胺能神经元或人神经母细胞瘤SH-SY5Y分化得到的人多巴胺能神经元;骨骼肌细胞腔室(3)培养的细胞为人骨骼肌细胞HSKMC(Human skeletal muscle cells)。
值得说明的是,本发明芯片内细胞均为人源的细胞。
更进一步的,经由所述外源炎症因子通道(6)加入的外源炎症因子包括天然或人工黑色素、蛋白质、小分子药物等一种或多种的组合。
本发明基于帕金森病已经阐明的机制之一:即中脑黑质区域病变导致小胶质细胞非正常地暴露于神经黑色素中,使得小胶质细胞被激活而引发神经炎症,从而损伤多巴胺能神经元,导致多巴胺合成的受阻,宏观上引起肌肉强直,震颤麻痹等症状。本发明中,外源的炎症因子加入诱导小胶质细胞的炎症发生,炎症因子随即扩散到相邻的神经元腔室引发神经元损伤,即神经炎症的产生;另一侧,神经元与骨骼肌细胞连通形成神经-肌肉接头,炎症导致的神经元损伤发生后,骨骼肌细胞会产生异常放电等现象,以上都与帕金森病灶微环境相关细胞的行为具有原理和现象上的相似性。因此,本发明公开的含细胞的-体外模拟神经炎症/帕金森病灶的微流控芯片有效地重现了神经炎症导致的帕金森病发生过程和相关的级联反应。
进一步的,所述细胞培养腔室的宽度为0.5cm-2cm,长度为0.3cm-1cm;所述微柱阵列(7)由边长0.003-0.005cm、顶角60°的菱形微柱组成;所述菱形微柱的间隔为0.003-0.005cm。
值得说明的是,所述培养基入口通道(4)用于连接数控蠕动泵控制的注射器,操控芯片内的培养基流动,控制推进速度为10-50μL/min。
值得说明的是,本发明所述的微流控芯片分为上下两层。上层为通道层,包括并列连接的小胶质细胞腔室(1)、神经元腔室(2)和骨骼肌细胞腔室(3),每个细胞培养腔室两侧分别设置培养基入口通道(4)和废液出口通道(5);小胶质细胞腔室(1)与外源炎症因子通道(6)相联通;相邻细胞培养腔室间由微柱阵列(7)相隔。所述通道层各部分由透明的聚合物材料制成,所述聚合物材料包括硅橡胶、天然橡胶、聚苯乙烯、聚乙烯、石英玻璃或有机玻璃。下层为基底层,由平整的硅橡胶或石英玻璃制成,上下两层经等离子体处理后键合。
本发明的第二个目的是提供如上所述的含细胞的微流控芯片的制备方法。
一种如上所述的含细胞的微流控芯片的制备方法,包括以下步骤:
步骤I:通过微加工表面刻蚀技术按照设计在硅片上制备带有图案的模板,后将PDMS预聚物浇筑于模板表面并固化,脱模、打孔后得到包括小胶质细胞腔室(1)、神经元腔室(2)、骨骼肌细胞腔室(3)、培养基入口通道(4)、废液出口通道(5)、外源炎症因子通道(6)和微柱阵列(7)的通道层;
步骤II:在平整的硅片表面旋涂PDMS预聚物,加热固化后得到硅橡胶下层基底,或直接取平整的石英玻璃作为基底层;
步骤III:将步骤I得到的通道层和步骤II得到的基底层进行等离子体处理,键合组装,得到微流控芯片原型;
步骤IV:首先将人神经元前体细胞接种于神经元腔室(2),加入分化培养基并待前体细胞分化为神经元细胞后,向小胶质细胞腔室(1)和骨骼肌细胞腔室(3)接种小胶质细胞和骨骼肌细胞;
步骤V:待微流控芯片原型内细胞状态稳定且神经-肌肉接头建立后,由外源炎症因子通道(6)加入外源炎症因子;并由各培养基入口通道(4)注入培养基以外源炎症因子的输入和培养基循环构建,模拟病灶微环境,得到含细胞的微流控芯片。
值得说明的是,本发明所公开的微流控芯片原型和含细胞的微流控芯片的区别在于,微流控芯片原型指的是通过上述步骤I~III制备得到,而未在内部接种和培养细胞的空白芯片。
更进一步的,本发明所公开的细胞培养基为MEM/F12或DMEM/F12完全培养基,具体成分是,MEM/F12或DMEM/F12+15%FBS+1%P/S。
更进一步的,所述微流控芯片原型与细胞接触的表面在步骤IV细胞接种前进行生物活性预处理。
本发明的第三个目的是提供如上所述的含细胞的微流控芯片的应用。
为了实现上述目的,本发明采用如下技术方案:
一种含细胞的微流控芯片在体外模拟神经炎症/帕金森病灶、基因和蛋白组学、细胞-药物相互组用评价、药效评价、药物渗透性、神经-肌肉电生理学和帕金森病新药筛选领域的应用。
值得说明的是,疾病模型是帕金森病研究的重要工具和手段,虽然目前已有以大鼠为实验动物通过注射方法构建多巴胺能神经元急性死亡的帕金森病模型构建方法,但是这些动物模型无法展示多巴胺的神经元的进行性和选择性损失,且也不会产生帕金森病患者特征性的路易体蛋白质块。因此,在体外构建人源细胞的帕金森疾病模型对更精准深入地研究帕金森病及开发相关药物具有重要意义。本发明首次设计了基于神经黑色素引发神经炎症的体外帕金森病灶微流控平台,通过腔室的设计和细胞株的选择,在易于构建的同时能够直观反应帕金森病灶部位的细胞行为和相关的级联反应。由此,本发明了一种基于人源细胞的高保真帕金森病灶环境及神经炎症发生过程的构建策略,并将其应用于帕金森病相关的基因和蛋白组学、药物评价与筛选相关的研究。
进一步的,所述应用为通过具有连续拍摄功能的活细胞工作站观察神经元腔室(2)中的多巴胺能神经元的程序性凋亡。
更进一步的,所述应用还包括在多巴胺能神经元程序性凋亡发生后,通过线粒体膜电位检测试剂盒测定骨骼肌细胞腔室(3)中骨骼肌细胞的膜电位变化。
更进一步的,所述应用还包括在多巴胺能神经元程序性凋亡发生后,向神经元腔室(2)注入左旋多巴、多巴胺受体激动剂等帕金森药物,观察和评价药物作用下的细胞反应和作用效果。
与现有技术相比,本发明优点在于:
1)本发明采取微流控技术公开了含细胞的微流控芯片,用于体外神经炎症/帕金森病灶模型的构建,将神经炎症的发生与帕金森病灶部位微环境结合,实现了帕金森病进行性发病过程的体外复现。
2)依托本发明提供的技术方案,有效整合了神经炎症/帕金森病发生状态下的细胞或局部组织的动态变化过程,与传统的动物建模相比,该模型能够满足细胞行为的连续实时观测和便捷的分子生物学检测要求,更适用于帕金森病高通量药物筛选和基因蛋白组学研究。
3)本发明构建的微流控模型芯片可重复性好,易于制作;采取商业化的人源细胞株,在构建高保真度模型,重现人患病细胞及组织区别于非灵长类动物的特异性表现的同时,避免了以动物为建模对象导致的成本问题和伦理争议。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1为本发明实施例1中制备的含细胞的微流控芯片主视图,图中标号为:1、小胶质细胞腔室;2神经元腔室;3、骨骼肌细胞腔室;4、培养基入口通道;5、废液出口通道;6、外源炎症因子通道;7、微柱阵列。
图2为本发明实施例1中腔室间微柱阵列的细节结构示意图和扫描电镜拍摄的实物图。
图3为本发明实施例1公开的微流控芯片原型的制备方法示意图。
图4为本发明实施例4中在含细胞的微流控芯片中研究黑色素对小胶质细胞的炎症激活效应的检测结果。
图5为本发明实施例5中在含细胞的微流控芯片构建帕金森疾病模型的芯片实物图及示意图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
在这里专用的词“实施例”,作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。本申请实施例中性能指标测试,除非特别说明,采用本领域常规试验方法。应理解,本申请中所述的术语仅仅是为描述特别的实施方式,并非用于限制本申请公开的内容。
除非另有说明,否则本文使用的技术和科学术语具有本申请所属技术领域的普通技术人员通常理解的相同含义;作为本申请中其它未特别注明的试验方法和技术手段均指本领域内普通技术人员通常采用的实验方法和技术手段。
在本发明的描述中,需要理解的是,术语“中”、“上”、“下”、“升”、“降”、“竖直”、“面”、“顶”、“底”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的设备或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制。
为了更好的说明本申请内容,在下文的具体实施例中给出了众多的具体细节。本领域技术人员应当理解,没有某些具体细节,本申请同样可以实施。在实施例中,对于本领域技术人员熟知的一些方法、手段、仪器、设备等未作详细描述,以便凸显本申请的主旨。
在不冲突的前提下,本申请实施例公开的技术特征可以任意组合,得到的技术方案属于本申请实施例公开的内容。
为更好地理解本发明,下面通过以下实施例对本发明作进一步具体的阐述,但不可理解为对本发明的限定,对于本领域的技术人员根据上述发明内容所作的一些非本质的改进与调整,也视为落在本发明的保护范围内。
实施例1微流控芯片原型的制备方法
(1)掩模板的制作
利用计算机绘图软件(CAD)设计并绘制微流控芯片内部的组织结构。借助高精度制板机制备掩模板。
(2)SU-8模板的加工
取3英寸单抛硅片于Piranha溶液(98%浓硫酸:30%过氧化氢溶液为3:1体积比)中预处理,用超纯水清洗3次后氮气吹干,置入真空烘箱中备用;对上述处理后的硅片用六甲基二硅氧烷进行气相表面处理。取3g SU-8负性光刻胶旋涂于上述硅片表面,形成约300μm厚的胶层后梯度升温至95℃软烤。用事先加工好的掩模板紧贴于旋涂完光刻胶的硅片上,在高强度紫外光下进行曝光。而后梯度升温至95℃硬烘。完成后,缓慢降至室温,用负性光刻胶显影液浸洗硅片以除去未曝光的SU-8光刻胶,在完全除去后依次用异丙醇、超纯水淋洗,用氮气吹干表面,留待后续使用。
(3)微流控芯片细胞培养腔室与流体通道层的制作
取适量的PDMS预聚物和交联剂(Dow corning 184)以10:1的质量比混合后脱除气泡,浇筑在SU-8模板上,在70℃烘胶台上加热固化12h。将固化后的PDMS从模板上剥离下来,最后用打孔器在流体通道两端对应位置打孔。
(4)细胞黏附底层的制作
对于PDMS细胞黏附底层,取适量的PDMS预聚物和交联剂(Dow corning184)以10:1的质量比混合后脱除气泡,浇筑在预先用六甲基二硅氧烷进行气相表面处理过的硅片上旋涂,在70℃烘胶台上加热2h后固化。其他材料的底膜层直接裁取与芯片上层尺寸相近的材料即可。
(5)微流控芯片的组装键合
将上述两个步骤中得到的芯片上层和细胞黏附底层一同置于等离子体仪中对表面进行氧等离子体处理,完毕后将两层对接、压紧,置于70℃烘胶台上加热固化后,刻划芯片边缘,键合后的芯片即为用于后续细胞培养的微流控芯片原型。
制备得到的微流控芯片原型结构如图1所示,腔室间微柱阵列的细节结构示意图和扫描电镜拍摄的实物图如图2所示,微流控芯片的制备方法示意图如图3所示。
实施例2含细胞的微流控芯片的芯片处理与细胞培养
(1)微流控芯片原型内表面的生物活性预处理
首先对微流控芯片原型进行灭菌处理,具体步骤如下:将微流控芯片原型置于紫外光下照射30min,后依次向通道内通入75%酒精和灭菌PBS;随后向通道内注入0.1mg/mL多聚赖氨酸在37℃孵育过夜,用灭菌PBS和完全培养基冲洗3次。
(2)细胞接种
取预先在细胞培养瓶中培养至80%汇合的细胞,用胰蛋白酶消化后离心收集细胞,以4×10^5个/mL的细胞密度将细胞悬液经由培养基入口通道注入到微流控芯片的细胞培养腔室中。细胞接种完毕后,将芯片置入培养皿放入细胞培养箱(温度为37℃,二氧化碳浓度为5%,氧气浓度为20%)中过夜使细胞能够稳定贴壁生长。细胞稳定黏附后将培养基入口通道与电动蠕动流体控制芯片相连,培养基出口通道与废液收集管相连,持续为芯片内提供新鲜的培养基置换,以确保细胞在芯片内的正常生长。
(3)细胞观察
开启并预热活细胞工作站(Carl Zeiss,兼具活细胞培养和倒置荧光显微镜拍摄功能),设定载物台培养温度为37℃,二氧化碳浓度为5%,氧气浓度为20%,待稳定后,将预先在培养箱中培养的微流控芯片转移至载物台上,利用显微镜自带软件选取观察点,设定持续时间和拍摄间隔,对芯片内细胞的进行连续拍摄。根据需要,在拍摄结束后,对芯片内的细胞用多聚甲醛固定,随后进行免疫荧光染色,在显微镜荧光模式下进行拍摄。
(4)神经元分化
对于人骨髓干细胞衍生的MNs的分化,首先使细胞在多聚赖氨酸预涂覆的表面上稳定黏附,换用含有10ng/mL碱性成纤维细胞生长因子的NB2培养基中培养至80%汇合后消化重新接种,先在启动培养基中培养4天,再转用分化培养基培养6天,得到人运动神经元。
对于人多巴胺能神经元前体细胞LUHMES的分化,首先使细胞在多聚赖氨酸预涂覆的表面上稳定黏附,随后换用分化培养基中分化48小时后撤去,换用基础培养基再继续分化48小时得到人多巴胺能神经元。
对于人神经母细胞瘤SH-SY5Y的分化,首先使细胞在多聚赖氨酸预涂覆的表面上稳定黏附,换用含有视黄醇酸的完全培养基作为分化培养基处理5-7天后撤去,换用含有脑源性神经营养因子BDNF的无血清培养基继续处理5天后分化得到人多巴胺能神经元。
实施例3含细胞的微流控芯片内的细胞蛋白质检测
(1)制作实施例1中相同的微流控芯片原型。
(2)按照实施例2中相同的方法将细胞接种在微流控芯片原型内,培养芯片在与电动蠕动流体控制芯片相连有连续培养基供给的情况下,培养7天-3周。
(3)通过培养基出口通道收集培养基。使用透析法、冷冻干燥、凝胶浓缩等方法,浓缩培养基中的蛋白质。使用BCA蛋白质浓度测定试剂盒定量蛋白质浓度。然后借助蛋白质免疫印迹法(Westernblot)分析蛋白质的表达情况。
实施例4黑色素对小胶质细胞的炎症激活效应研究
(1)制作实施例1中相同的微流控芯片原型,其中细胞黏附底层采用纤连蛋白对基底层进行预先处理后与通道层键合。
(2)按照实施例2中相同的方法将小胶质细胞株HMC3接种在微流控芯片原型内,培养芯片在与电动蠕动流体控制芯片相连有连续培养基供给的情况下,培养7天-3周。
(3)培养24小时使得细胞稳定贴壁后,由外源炎症因子通道向培养人小胶质细胞的培养腔室输入不同浓度的神经黑色素。
(4)向通道内注入预冷的细胞裂解液,将芯片内的细胞裂解完全,离心,收集上清液得到蛋白质。使用BCA蛋白质浓度测定试剂盒定量蛋白质浓度。然后借助蛋白质免疫印迹法(Western blot)分析蛋白质的表达情况,结果如图4所示。
实施例5帕金森体外病灶模型的构建
(1)制作实施例1中相同的微流控芯片原型。
(2)对芯片内表面进行生物活性处理:向各通道内注入纤连蛋白溶液在0℃保持24h或用0.1mg/mL的多聚赖氨酸溶液在37℃培养箱中孵育过夜,随后去除涂覆溶液并用PBS冲洗通道;
(3)按照实施例2中相同的方法将细胞接种在芯片内。具体地,在芯片内进行神经元分化前体细胞的接种并按照实施例2中的步骤完成分化过程,得到人神经元细胞(包括由人骨髓干细胞衍生的MNs分化得到的人运动神经元和由永生化多巴胺能神经元前体细胞LUHMES或人神经母细胞瘤SH-SY5Y分化而来的多巴胺能神经元),随后向芯片内接种人小胶质细胞HMC3和人骨骼肌细胞HSKMC(Human Skeletal Muscle Cell)。细胞稳定黏附后,借助电动蠕动流体控制芯片从培养基入口通道向芯片内持续通入MEM(Minimum EssentialMedium,含NEAA)完全培养基。细胞稳定黏附后,由外源炎症因子通道向培养人小胶质细胞的培养腔室输入神经黑色素,根据实施例4中的结果,优选地,可采取的神经黑色素浓度为0.05mg/mL,培养周期为7天至4周。
所述含细胞的微流控芯片的具体结构如图5。
在培养过程中,神经黑色素引发了小胶质细胞的炎症反应,导致了多巴胺能神经元细胞的进行性损伤和死亡,这与帕金森发病过程中的多巴胺能神经元进行性死亡过程具有机理上的一致性。从而说明本发明中的局部脑组织微流控芯片可以方便简易地模拟帕金森病的进行性发病过程。同时,运动神经元与骨骼肌细胞通过腔室间微柱阵列形成神经-肌肉接头,神经元发生进行性死亡的过程中,肌肉细胞的电生理信号和形态也会发生相应变化,这与帕金森病发生引起的肌肉僵直等症状具有现象上的一致性。
实施例6利用神经黑色素引发炎症的神经炎症模型的构建
(1)制作实施例1中相同的初始微流控芯片(或称“微流控芯片原型”),含细胞的微流控芯片的具体结构如图5。
(2)按照实施例2中相同的方法将细胞接种在初始微流控芯片内。具体地,将人永生化的小胶质细胞HMC3(Human Microglia Clone 3)接种在芯片内待其稳定黏附后,借助电动蠕动流体控制芯片从培养基入口通道向芯片内持续通入MEM(Minimum EssentialMedium,含NEAA)完全培养基。之后,由外源炎症因子通道向腔室内输入神经黑色素,根据实施例4中的结果,优选地,可采取的神经黑色素浓度为0.05mg/mL。
实施例7利用外源糖蛋白引发炎症的神经炎症模型的构建
(1)按照实施例6中相同的方法构建微流控芯片原型,将人永生化的小胶质细胞HMC3(Human Microglia Clone 3)接种在芯片内,待其稳定黏附后利用电动蠕动流体控制芯片向芯片内通入完全培养基。
(2)由外源炎症因子通道向腔室内输入干扰素γ-IFN,优选地,可采取的干扰素γ-IFN浓度为5-10ng/mL。
实施例8、利用多种外源炎症因子引发小胶质细胞的神经炎症
(1)按照实施例6中相同的方法构建微流控芯片原型,将人永生化的小胶质细胞HMC3(Human Microglia Clone 3)接种在芯片内,待其稳定黏附后利用电动蠕动流体控制芯片向芯片内通入完全培养基。
(2)由外源炎症因子通道向腔室内输入合成神经黑色素和天然神经黑色素的混合物,优选地,两神经黑色素的终浓度均为0.01-0.025mg/mL。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种含细胞的微流控芯片,其特征在于,所述微流控芯片依次包括三个并列的细胞培养腔室:小胶质细胞腔室(1)、神经元腔室(2)和骨骼肌细胞腔室(3),且所述神经元腔室(2)位于小胶质细胞腔室(1)和骨骼肌细胞腔室(3)之间;每个细胞培养腔室两侧分别设置培养基入口通道(4)和废液出口通道(5);小胶质细胞腔室(1)与外源炎症因子通道(6)相联通;相邻细胞培养腔室间由微柱阵列(7)相隔。
2.根据权利要求1所述的微流控芯片,其特征在于,所述小胶质细胞腔室(1)培养的细胞为人永生化小胶质细胞株HMC3;神经元腔室(2)培养的细胞为经人骨髓干细胞衍生的MNs分化得到的人运动神经元、经人多巴胺能神经元前体细胞LUHMES分化得到的人多巴胺能神经元或人神经母细胞瘤SH-SY5Y分化得到的人多巴胺能神经元;骨骼肌细胞腔室(3)培养的细胞为人骨骼肌细胞HSKMC。
3.根据权利要求2所述的微流控芯片,其特征在于,经由所述外源炎症因子通道(6)加入的外源炎症因子包括天然或人工黑色素、蛋白质、小分子药物等一种或多种的组合。
4.根据权利要求1所述的微流控芯片,其特征在于,所述细胞培养腔室的宽度为0.5cm-2cm,长度为0.3cm-1cm;所述微柱阵列(7)由边长0.003-0.005cm,顶角60°的菱形微柱组成;所述菱形微柱的间隔为0.003-0.005cm。
5.一种如权利要求1-4任一项所述的含细胞的微流控芯片的制备方法,其特征在于,包括以下步骤:
步骤I:通过微加工表面刻蚀技术按照设计在硅片上制备带有图案的模板,后将PDMS预聚物浇筑于模板表面并固化,脱模、打孔后得到包括小胶质细胞腔室(1)、神经元腔室(2)、骨骼肌细胞腔室(3)、培养基入口通道(4)、废液出口通道(5)、外源炎症因子通道(6)和微柱阵列(7)的通道层;
步骤II:在平整的硅片表面旋涂PDMS预聚物,加热固化后得到硅橡胶下层基底,或直接取平整的石英玻璃作为基底层;
步骤III:将步骤I得到的通道层和步骤II得到的基底层进行等离子体处理,键合组装,得到微流控芯片原型;
步骤IV:首先将人神经元前体细胞接种于神经元腔室(2),加入分化培养基并待前体细胞分化为神经元细胞后,向小胶质细胞腔室(1)和骨骼肌细胞腔室(3)接种小胶质细胞和骨骼肌细胞;
步骤V:待微流控芯片原型内细胞状态稳定且神经-肌肉接头建立后,由外源炎症因子通道(6)加入外源炎症因子;并由各培养基入口通道(4)注入培养基以外源炎症因子的输入和培养基循环构建,模拟病灶微环境,得到含细胞的微流控芯片。
6.根据权利要求5所述的微流控芯片的制备方法,其特征在于,微流控芯片原型与细胞接触的表面在步骤IV细胞接种前进行生物活性预处理。
7.一种使用如权利要求1~4任一项所述的含细胞的微流控芯片的应用,其特征在于,所述含细胞的微流控芯片在体外模拟神经炎症/帕金森病灶、基因和蛋白组学、细胞-药物相互组用评价、药效评价、药物渗透性、神经-肌肉电生理学和帕金森病新药筛选领域的应用。
8.根据权利要求7所述的应用,其特征在于,通过具有连续拍摄功能的活细胞工作站观察神经元腔室(2)中的多巴胺能神经元的程序性凋亡。
9.根据权利要求8所述的应用,其特征在于,在多巴胺能神经元程序性凋亡发生后,通过线粒体膜电位检测试剂盒测定骨骼肌细胞腔室(3)中骨骼肌细胞的膜电位变化。
10.根据权利要求8或9任一项所述的应用,其特征在于,在多巴胺能神经元程序性凋亡发生后,向神经元腔室(2)注入左旋多巴、多巴胺受体激动剂等帕金森药物,观察和评价药物作用下的细胞反应和作用效果。
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