CN106811411B - 一种基于微流控芯片的人心脏模型的建立方法 - Google Patents

一种基于微流控芯片的人心脏模型的建立方法 Download PDF

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CN106811411B
CN106811411B CN201510869868.XA CN201510869868A CN106811411B CN 106811411 B CN106811411 B CN 106811411B CN 201510869868 A CN201510869868 A CN 201510869868A CN 106811411 B CN106811411 B CN 106811411B
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秦建华
徐聪
于跃
王丽
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Abstract

本发明提供一种基于微流控芯片的人心脏模型的建立方法。该微流控芯片主要由细胞入口池、通道、细胞培养室和出口池组成，细胞培养室经直通道连接细胞入口池和出口池，细胞培养室为带有重复六边形凸起结构。人心脏模型的建立方法，按照以下步骤进行：(1)hiPSC定向分化成心肌细胞；(2)芯片修饰；(3)芯片内细胞的接种与培养；(4)人心脏模型的建立。该模型用于观察芯片内细胞活性考察及功能变化的表征；可用于人心脏发育学研究以及药物心脏毒性评价。本发明所述的一种基于微流控芯片的人心脏模型，特别针对心脏发育过程中，血流剪切力和基质表面微结构对心肌细胞发育成熟度的影响，观察心肌细胞在带有微结构表面以及不同流速灌流条件下的形态变化和排列。

Description

一种基于微流控芯片的人心脏模型的建立方法
技术领域
本发明属微流控芯片技术应用到组织仿生以及实时监测的细胞生物学研究的技术领域，具体涉及一种基于微流控芯片的人心脏模型的建立方法。
背景技术
心血管疾病是世界头号死因，每年死于心血管疾病的人数多于其他任何疾病。在药物研发过程中，超过1/3的药物由于心脏毒性而无法通过安全测试而被撤回。药物进行临床实验前需要进行大量的动物实验，这些实验一般十分昂贵，耗时长，伦理争议大，而且由于种属差异，不能准确反映药物在人体内的作用，往往导致药物在临床试验中的失败。传统二维细胞培养模型由于无法模拟器官在体内的微环境，功能不全，对药物的反应也与体内情况相差巨大。因此，亟需一种有效的体外心脏模型用于药物研发和毒性检测。
器官芯片是一种在微米级小室中连续培养组织细胞的微流控装置，它通过调节细胞的排布，微通道的设计等模拟组织微环境，能很好地再现组织或器官水平的生理活动和功能，为药物研发提供体外模型。人诱导多能干细胞(hiPSCs)的出现，为建立人体生理病理模型提供了可能。通过将hiPSCs定向诱导分化成不同的细胞类型，科学家构建了多种器官芯片模型，如肺，肝，血管，心脏等，并成功应用于药物筛选和毒理性研究。
HiPSCs诱导获得的心肌细胞往往呈现出胎儿期的特性，其形态，结构和功能都不成熟，作为药物筛选模型具有一定风险。体外心肌细胞的成熟与培养时间，流体剪切力，基质软硬度，细胞排列图案，电刺激等物理化学因素密切相关。因此，可以利用微流控技术在芯片上模拟这些物理化学因素，促进hiPSCs来源的心肌细胞成熟，建立更符合成年人心脏功能指标的体外心脏模型，为药物研发与筛选提供技术平台。
发明内容
本发明的目的是提供一种基于微流控芯片的人心脏模型的建立方法，并应用于药物心脏毒性评价。
一种微流控芯片，该微流控芯片主要由细胞入口池、通道、细胞培养室、通道和出口池组成，细胞培养室经直通道连接细胞入口池和出口池。
该微流控芯片由上下两层芯片材料不可逆封接而成，上层芯片材料为带有腔室和通道的可透光透气的PDMS聚合物，其上具有覆盖基底表面图案的腔室以及通道；
下层芯片材料为可透光透气的，表面带有重复六边形凸起结构的PDMS聚合物，表面带有重复六边形凸起结构的PDMS聚合物；
上层芯片材料经等离子体处理30-60s不可逆封接于下层材料上，上层芯片的细胞培养室覆盖基底带有结构的区域。
上层芯片材料的腔室及通道高度为200-2000um，可适用于流速为0.1ul/min-100ul/min的灌流培养。
下层基底材料为六边形凸起的六边形长30-600um，宽10-100um，间距5-40um，高2-200um，重复个数20-2000。
本发明还提供了一种基于微流控芯片的人心脏模型的建立方法，采用上述微流控芯片，基本过程如下：
(1)hiPSC定向分化成心肌细胞
将人诱导多能干细胞接种到Matrigel包被的平板上培养至细胞密度增至孔板底面积的80％-90％，更换为RPMI 1640/B27-insulin培养基，并加入CHIR99021(终浓度12uM)处理24小时，然后换成RPMI1640/B27-insulin培养基培养，2天后加入IWP-2(终浓度5uM)培养2天，最后更换为RPMI 1640/B27培养基，每2-3天换一次培养基。
(2)芯片修饰
用移液器将配制好的Matrigel工作液经由细胞入口池加入芯片，将固定芯片的培养皿4℃静置过夜，12-36小时(优选24小时)后37℃孵育30-90min(优选60min)，对通道底面进行修饰，促进人诱导多能干细胞来源的心肌细胞(HiPSC-CM)更好地贴壁；
(3)芯片内细胞的接种与培养
诱导的心肌细胞出现跳动后一周，用酶消化后，调整至合适的细胞密度(细胞密度为5×106cells/mL)，经由细胞入口池加入到芯片中，在Matrigel的修饰作用下，细胞迅速贴壁并均匀铺展于细胞培养室底面，当在光学显微镜下观察到细胞在细胞培养室中均匀分布时，立即将芯片移入二氧化碳培养箱中继续培养；
(4)人心脏模型的建立
待HiPSC-CM细胞经过沉淀完全铺满细胞培养室底面后，经由细胞入口池向通道中通入不含血清的细胞培养液并继续培养3-10天，使HiPSC-CM细胞处于流体剪切力的作用下。
一种基于微流控芯片的人心脏模型的应用，用于人心脏模型的芯片内细胞活性考察及功能变化的表征，具体包括以下几个方面：
(1)显微镜观察心肌细胞形态和跳动情况；
(2)常规免疫荧光染色法表征流体剪切作用后的HiPSC-CM细胞内cTnT的表达，用于表征心肌细胞成熟过程中结构和功能的变化，主要表征细胞内骨架的变化。
(3)RT-PCR检测细胞NK2.5、MYH6、MYH7、hCX43等基因的相对表达水平，表征细胞功能在表达水平的变化。
本发明的芯片具有良好的生物相容性，可长时间培养心肌细胞。利用本芯片可实现心肌细胞的灌流培养，模拟在体血液流动情况，研究血液剪切力对心肌细胞功能的作用。细胞培养室的表面结构有利于心肌细胞在形态上的拉伸和对齐，促进心肌细胞成熟和功能。
本发明一种基于微流控芯片的人心脏模型，可用于人心脏发育学研究以及药物心脏毒性评价。特别针对心脏发育过程中，血流剪切力和基质表面微结构对心肌细胞发育成熟度的影响，观察心肌细胞在带有微结构表面以及不同流速灌流条件下的形态变化和排列。
附图说明
图1本发明微流控芯片整体结构示意图；
图2本发明微流控芯片通道内，hiPSC-CM细胞的贴壁与铺展情况；
图3灌流培养条件下，hiPSC-CM心肌细胞cTnT染色情况：心肌细胞沿凸起结构长轴方向拉伸并对齐，表现出成熟心肌细胞的部分形态特征。
其中：1细胞入口池、2通道、3细胞培养室和4出口池。
具体实施方式
下面的实施例将对本发明予以进一步的说明，但并不因此而限制本发明。
一种微流控芯片，结构如图1所示：该微流控芯片主要由细胞入口池1、通道2、细胞培养室3和出口池4组成，细胞培养室3经直通道2连接细胞入口池1和出口池4，细胞培养室3为带有重复六边形凸起结构。
该微流控芯片由上下两层芯片材料不可逆封接而成，上层芯片材料为带有腔室和通道的可透光透气的PDMS聚合物，其上具有覆盖基底表面图案的腔室以及通道；
下层芯片材料为可透光透气的，表面带有重复六边形凸起结构的PDMS聚合物，表面带有重复六边形凸起结构的PDMS聚合物；
上层芯片材料经等离子体处理30-60s不可逆封接于下层材料上，上层芯片的细胞培养室覆盖基底带有结构的区域。
上层芯片材料的腔室及通道高度为200-2000um，可适用于流速为0.1ul/min-100ul/min的灌流培养。
下层基底材料为六边形凸起的六边形长30-600um，宽10-100um，间距5-40um，高2-200um，重复个数20-2000。
实施例1
应用HhiPSC-CM细胞建立人心脏模型
利用上述微流控芯片，结构如图1所示。用DMEM-F12培养基配制浓度为150μg/mL的Matrigel工作液，经由细胞入口池注入芯片内，4℃静置过夜，24小时后移除工作液。将hiPSC-CM细胞消化后，稀释成浓度为5×106cells/mL的细胞悬液，经由细胞入口池加入100μL细胞悬液至芯片内，在Matrigel的修饰作用下，细胞迅速贴壁并均匀铺展于细胞培养室底面(图2)，当在光学显微镜下观察到细胞在细胞培养室中均匀分布时，立即将芯片移入二氧化碳培养箱中继续培养。待hiPSC-CM细胞完全贴附细胞培养室底面后，经由细胞入口池分别向通道中通入细胞培养液，设定流速为1ul/min，模拟心脏组织间隙流。
实施例2
人心脏模型细胞内功能蛋白的表征
利用实验室自行设计制作的微流控芯片，结构如图1所示。芯片修饰后，采用与实施例1相同的细胞接种和培养方式建立心脏模型。灌流7天后，进行细胞免疫荧光染色，检测蛋白为肌钙蛋白(cTnT)。方法如下：4％多聚甲醛进行细胞固定，PBS缓冲液冲洗三次，每次10min；0.1％triton X-100致孔剂作用10min，PBS缓冲液冲洗三次，每次10min；山羊封闭血清作用1h，一抗(小鼠抗人cTnT)1:100稀释，4℃孵育过夜，PBS缓冲液冲洗三次，每次10min；二抗(Alexa Fluor488标记的山羊抗小鼠IgG)1:100稀释，常温避光孵育1h，PBS缓冲液冲洗三次，每次10min；冲洗完毕后加入1:2000稀释的DAPI工作液孵育15min，PBS缓冲液冲洗2次，荧光显微镜下拍照，记录相应蛋白的表达情况，结果如图3所示。在权利要求的芯片上，心肌细胞沿着表面结构排列。

Claims (4)

1.一种用于建立人心脏模型的微流控芯片，其特征在于：该微流控芯片主要由细胞入口池(1)、通道(2)、细胞培养室(3)和出口池(4)组成，细胞培养室(3)经直通道(2)连接细胞入口池(1)和出口池(4)，细胞培养室(3)为带有重复六边形凸起结构；
该微流控芯片由上下两层芯片材料不可逆封接而成，上层芯片材料为带有腔室和通道的可透光透气的PDMS聚合物，其上具有覆盖基底表面图案的腔室以及通道；
下层芯片材料为可透光透气的，表面带有重复六边形凸起结构的PDMS聚合物；
上层芯片材料经等离子体处理30-60s不可逆封接于下层材料上，上层芯片的细胞培养室覆盖基底带有结构的区域；
上层芯片材料的腔室及通道高度为200-2000um，可适用于流速为0.1ul/min-100ul/min的灌流培养；
下层芯片材料为六边形凸起的六边形长30-600um，宽10-100um，间距5-40um，高2-200um，重复个数20-2000。
2.一种基于权利要求1所述的微流控芯片的人心脏模型的建立方法，其特征在于按照以下步骤进行：
(1)hiPSC定向分化成心肌细胞
将人诱导多能干细胞接种到Matrigel包被的平板上培养至细胞密度增至孔板底面积的80％-90％，更换为RPMI 1640/B27-insulin培养基，并加入CHIR99021处理24小时，CHIR99021终浓度为12uM，然后换成RPMI1640/B27-insulin培养基培养，2天后加入IWP-2培养2天，IWP-2的终浓度为5uM，最后更换为RPMI 1640/B27培养基，每2-3天换一次培养基；
(2)芯片修饰
用移液器将配制好的Matrigel工作液经由细胞入口池加入芯片，将固定芯片的培养皿4℃静置过夜，12-36小时后37℃孵育30-90min，对通道底面进行修饰，促进人诱导多能干细胞来源的心肌细胞(HiPSC-CM)更好地贴壁；
(3)芯片内细胞的接种与培养
诱导的心肌细胞出现跳动后一周，用酶消化后，调整细胞密度为5×106cells/mL，经由细胞入口池加入到芯片中，在Matrigel的修饰作用下，细胞迅速贴壁并均匀铺展于细胞培养室底面，当在光学显微镜下观察到细胞在细胞培养室中均匀分布时，立即将芯片移入二氧化碳培养箱中继续培养；
(4)人心脏模型的建立
待HiPSC-CM细胞经过沉淀完全铺满细胞培养室底面后，经由细胞入口池向通道中通入不含血清的细胞培养液并继续培养3-10天，使HiPSC-CM细胞处于流体剪切力的作用下。
3.按照权利要求2所述的微流控芯片的人心脏模型的建立方法，其特征在于步骤(2)中条件为：将固定芯片的培养皿4℃静置过夜，24小时后37℃孵育60min。
4.一种基于权利要求1所述的微流控芯片的人心脏模型的应用，其特征在于该模型用于观察芯片内细胞活性考察及功能变化的表征，具体包括以下几个方面：
(1)显微镜观察心肌细胞形态和跳动情况；
(2)常规免疫荧光染色法表征流体剪切作用后的HiPSC-CM细胞内cTnT的表达，用于表征心肌细胞成熟过程中结构和功能的变化，主要表征细胞内骨架的变化；
(3)RT-PCR检测细胞NK2.5、MYH6、MYH7、hCX43基因的相对表达水平，表征细胞功能在表达水平的变化。
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