CN114939446A - 一种平行通道微栅栏共培养多种细胞的微流控芯片及其应用 - Google Patents

一种平行通道微栅栏共培养多种细胞的微流控芯片及其应用 Download PDF

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CN114939446A
CN114939446A CN202210479303.0A CN202210479303A CN114939446A CN 114939446 A CN114939446 A CN 114939446A CN 202210479303 A CN202210479303 A CN 202210479303A CN 114939446 A CN114939446 A CN 114939446A
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罗勇
丛烨
高志刚
张秀莉
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Ningbo Research Institute of Dalian University of Technology
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Abstract

本发明属于微流控技术领域，一种平行通道微栅栏共培养多种细胞的微流控芯片及其应用。微流控芯片包括基板和基片，基板上包括流动培养通道和三维培养通道。流动培养通道的两端设置储液池，三维培养通道的两端设置三维培养通道进出液口。流动培养通道、与流动培养通道邻近的三维培养通道之间设置模拟内皮屏障的微栅栏结构，不同的三维培养通道之间设置三维培养通道间微栅栏。模拟内皮屏障的微栅栏结构与三维培养通道间微栅栏仅布置于流动培养通道和三维培养通道的底端。本发明将多种细胞分别平行接种在对应流体通道内，形成多种细胞的平行带状空间排布，为基于体外肝血窦模型的肝纤维化模型构建和相关研究提供工程化平台。

Description

一种平行通道微栅栏共培养多种细胞的微流控芯片及其应用
技术领域
本发明涉及一种平行通道微栅栏共培养多种细胞的微流控芯片及其应用，属于微流控技术领域。
背景技术
微流芯片技术是指通过使用特殊的几何结构(如微通道和微泵阀)精确控制微米和纳米级流体流动的技术。利用该技术，可以实现微升级别的生物，化学，医药等实际液体样品的分流、混合、提取、过滤和分析操作。该技术具有紧凑、快速、低耗和通量高的优点。随着微流控芯片技术的广泛应用，人们开始将其应用于细胞生物学和细胞分析领域，在技术上实现芯片上细胞培养、体内微环境模拟和构建、单细胞分析芯片，以及进一步的“器官芯片”模型构建。(Microfluidic organs-on-chips.Nat.Biotechnol.2014)
肝脏是人体内仅次于皮肤的第二大器官，从结构上看被分割成较大的右叶和较小的左叶，外部覆盖一层纤维组织。肝脏也是公认的多功能器官，目前研究认为其至少有500种不同的执行功能，包括血糖和氨水平的控制、各种激素的合成、铁和维生素的储存、以及内源性和外源性物质的解毒。肝脏由多种类型的常驻细胞组成，如肝实质细胞、肝星状细胞、枯否细胞和肝窦内皮细胞，这些细胞直接执行肝功能，并通过自分泌和旁分泌信号相互连接，共同形成复杂的信号传导和代谢环境。
肝脏的基本结构功能单位是肝小叶，肝小叶整体呈正六边形结构，正六边形的中心为中央静脉，肝板和肝血窦以中央静脉为中心放射状排列。肝板间的空隙称为肝血窦，血窦内皮与肝板之间的间隙称为窦间隙，窦间隙中充满着细胞外基质和少量的肝星状细胞，肝细胞血窦面上的微绒毛浸于其中，是肝细胞与血液之间进行物质交换的场所。
微制造和微流控技术的快速发展为在芯片上建立微尺度功能的肝脏提供了一种很有前途的方法。与传统培养物相比，微流控装置具有许多优势。例如，微流控装置可以很容易地产生浓度梯度，控制细胞的空间分布，并提供流动环境。詹森等人(Human livercell spheroids in extended perfusion bioreactor culture for repeated-dosedrug testing.Hepatology.2012)利用微流控生物反应器的优势获得维持长期肝功能的人肝细胞球状体。这些球状体在无血清条件下培养3-4周，维持其I期酶表达，并允许在长时间重复诱导。长期培养3-4周后，对人肝细胞球状体进行肝脏特异性标记物免疫荧光检测，可观察到肝细胞核因子、白蛋白、细胞角蛋白18和细胞色素P4503A的存在。然而，球状培养的细胞无序排布和中心缺氧坏死限制了其在体外模型中的应用。亚伯拉罕等人(Liversinusoid on a chip:Long-term layered co-culture of primary rat hepatocytesand endothelial cells in microfluidic platforms.Biotechnol.Bioeng.2015)构建了一种逐层叠加式的肝血窦芯片模型，利用注射泵提供双通道流体剪切力，多孔膜结构作为依托，结合芯片结构使细胞形成近生理的空间排布，培养原代大鼠肝细胞及内皮细胞单层模拟肝血窦结构，可实现30天长期培养。但这类技术使用的细胞种类较少，培养方式单一、受重力影响较大且存在对低通量、稳定性差和对大型泵阀设备依赖的缺陷。
发明内容
为了适应原位监测和逐细胞施加刺激的需求，解决逐层叠加式芯片受重力影响、细胞培养中心区域易缺氧的问题，本发明目的是提供一种平行通道微栅栏共培养多种细胞的微流控芯片及其应用。该微流控芯片引入了微栅栏结构将细胞按生理空间位置培养在几个平行的通道内，可以代替多孔膜提供空间隔断，同时可控的栅栏间隙利于加强芯片中的氧气及物质传输。该微流控芯片利用无泵驱动的流体流动方式，将多种细胞分别平行接种在对应流体通道内，形成多种细胞的平行带状空间排布，为基于体外肝血窦模型的肝纤维化模型构建和相关研究提供工程化平台。
为了实现上述发明目的，本发明采取的技术方案是：
一种平行通道微栅栏共培养多种细胞的微流控芯片，所述的微流控芯片包括一层基板和一层基片，所述的基板上包括流动培养通道和三维培养通道。所述的流动培养通道的两端设置储液池，三维培养通道的两端设置三维培养通道进出液口。流动培养通道、与流动培养通道邻近的三维培养通道之间设置模拟内皮屏障的微栅栏结构，不同的三维培养通道之间设置三维培养通道间微栅栏，作为物质交换和细胞迁移的通道。
所述的模拟内皮屏障的微栅栏结构与三维培养通道间微栅栏仅布置于流动培养通道和三维培养通道的底端。
优选的，所述的模拟内皮屏障的微栅栏结构与三维培养通道间微栅栏的设置高度为流动培养通道和三维培养通道高度的1/40
所述基片的材料选自石英、玻璃、PMMA或聚碳酸酯中的一种。
所述基板的材料选自石英、玻璃、PMMA、PDMS聚合物、聚酯、硅胶、琼脂糖、壳聚糖或海藻酸钠中的一种。
所述微流控芯片，在模拟肝血窦内多种肝细胞空间排布方面中的应用、在模拟血脑屏障结构内多种神经细胞空间排布方面的应用、在模拟肠道屏障功能中相关肠道细胞空间排布方面的应用、在模拟肾单位结构中多种肾脏细胞空间排布方面的应用。
当所述微流控芯片应用于模拟肝血窦内多种肝细胞空间排布方面时，所述流动培养通道和三维培养通道内通入的细胞根据需求，选自人、任何一种动物的肝窦内皮细胞、肝星状细胞、肝实质细胞和枯否细胞衍生的细胞系，或选自来源于人、任何一种动物的肝相关原代细胞，或使用iPS诱导分化的肝相关功能细胞的一种或多种。
优选的，所述微流控芯片设置一条流动培养通道和两条三维培养通道；所述流动培养通道中接种肝血窦内皮细胞，与流动培养通道邻近的三维培养通道进液口通入混有肝星状细胞的三维胶，另一条三维培养通道进液口通入混有肝实质细胞的三维胶，所述流动培养通道通入枯否细胞静置后，脱色摇床无泵驱动循环市售肝细胞生长完全培养基，两条三维培养通道灌满无血清的市售肝细胞完全培养基。
所述三维胶选自BME、Matrixgel或海藻酸钠胶中的一种。
本发明的有益效果是：所本发明的微流控芯片是一种加工一体化、结构简单、稳定性高并且可多次使用的平行通道微栅栏肝血窦芯片模型。结合二维、三维培养方式，模拟了肝窦状间隙的近生理细胞排布和微环境，采用无泵驱动的开放式单通道流动运行，为体外血流内皮屏障提供流体剪切力。利用该芯片构建的体外肝脏模型，能够实现基本肝功能、疾病研究、药物活性筛选研究的目的。
附图说明
图1是本实施例一种平行通道微栅栏共培养4种肝细胞的微流控芯片结构示意图。
图中：1、基片，2、基板，2a、流动培养通道，2b、第一三维培养通道，2c、第二三维培养通道，2d、储液池，2e、第一三维培养通道进出液口，2f、第二三维培养通道进出液口，2g、模拟内皮屏障的微栅栏结构，2h、三维培养通道间微栅栏。
图2是实施例1中肝血窦芯片接种肝窦内皮细胞衍生细胞系HUVEC细胞、枯否细胞衍生细胞系U937细胞、肝星状细胞衍生细胞系LX-2细胞和肝实质细胞衍生细胞系HepG2细胞后的效果图。
图3是实施例2中乙醛诱导的酒精性肝损伤检测的结果图。
图4是实施例3中脂多糖诱导的肝纤维化检测的结果图。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1
如图1所示，一种平行通道微栅栏共培养4种肝细胞的微流控芯片，所述微流控芯片整体尺寸为50mm*30mm*10mm，包括一层PMMA加工的基片1、一层PMMA加工的基板2，所述基板2包含一条流动培养通道2a，第一三维培养通道2b、第二三维培通道2c、储液池2d、第一三维培养通道进出液口2e、第二三维培养通道进出液口2f、模拟内皮屏障的微栅栏结构2g和三维培养通道间微栅栏2h，流动培养通道2a尺寸为3mm*20mm，第一三维培养通道2b、第二三维培通道2c尺寸均为1.5mm*20mm，其中流动培养通道2a两侧设有半径为5mm高度为8mm的圆形储液池2d，第一三维培养通道2b、第二三维培通道2c两侧分别设有半径为1.5mm的第一三维培养通道进出液口2e、第二三维培养通道进出液口2f，所述流动培养通道2a与第一三维培养通道2b间设置有长3.5mm，宽150μm，间隔150μm的模拟内皮屏障的微栅栏结构2g，所述第一三维培养通道2b、第二三维培通道2c之间设有长1mm，宽150μm，间隔150μm的两条三维培养通道间微栅栏2h用于物质交换和信息交流；如图2所示，所述流动培养通道2a内接种HUVEC细胞和附着在表面的U937细胞，所述第一三维培养通道2b内加入混有Matrixgel的LX-2细胞，所述第二三维培养通道2c内加入混有Matrixgel的HepG2细胞，所述第一三维培养通道2b、第二三维培通道2c内混有Matrixgel的细胞静置后，从第一三维培养通道进出液口2e、第二三维培养通道进出液口2f灌入无血清的市售HepG2细胞生长完全培养基，所述流动培养通道两侧储液池2d中循环市售含血清HepG2细胞培养基。
实施例2
本实施例采用实施例1中的微流控芯片模型，以乙醛诱导的酒精性肝损伤为例，微流控芯片运行正常以后，向第二三维培养通道2c内加入浓度为425μM的乙醛48h，每24h检测第二三维培养通道2c上清液中尿素氮含量，流动培养通道2a上清液中白蛋白含量，每48h检测芯片流动培养通道2a、第一三维培养通道2b、第二三维培通道2c上清液中一型前胶原和基质金属蛋白酶抑制剂含量，表征基于乙醛诱导的酒精性肝损伤模型，结果如图3所示，在乙醛的暴露作用下，微流控体外肝模型代谢和排泄功能下降，且分泌大量一型前胶原和基质金属蛋白酶抑制剂。
实施例3
本实施例采用实施例1中的微流控芯片模型，以脂多糖诱导的肝纤维化模型检测为例，微流控芯片运行正常以后，流动培养通道2a内循环含1μg/mL脂多糖的市售肝细胞完全培养基，每24h检测第二三维培养通道2c上清液中尿素氮含量，流动培养通道2a上清液中白蛋白含量，每48h检测芯片流动培养通道2a、第一三维培养通道2b、第二三维培养通道2c上清液中一型前胶原和基质金属蛋白酶抑制剂含量，表征基于脂多糖诱导的肝纤维化模型，结果如图4所示，在脂多糖诱导下，微流控体外肝模型代谢和排泄功能略下降，且分泌大量一型前胶原和基质金属蛋白酶抑制剂。
实施例4
本实施例采用实施例1中的微流控芯片模型，利用该微流控芯片模拟基于白介素6(IL-6)、转化生长因子β(TGF-β)和肿瘤坏死因子α(TNF-α)三种炎症配体激活终产物诱导的中后期肝纤维化模型。微流控芯片运行正常后，向流动培养通道2a循环包含浓度为100pg/mL TNF-α的市售肝细胞完全培养基，向第一三维培养通道2b加入包含浓度为5ng/mL TGF-β的无血清市售肝细胞培养基，向第二三维培养通道2c加入包含浓度为50pg/mL IL-6的无血清市售肝细胞培养基，每24h检测第二三维培养通道2c上清液中尿素氮含量，流动培养通道2a上清液中白蛋白含量，每48h检测芯片流动培养通道2a、第一三维培养通道2b、第二三维培养通道2c上清液中一型前胶原和基质金属蛋白酶抑制剂含量，表征基于多种炎症配体激活终产物诱导的中晚期肝纤维化损伤模型。
实施例5
本实施例采用实施例1中的微流控芯片模型，利用该微流控芯片评价基于乙醛诱导的酒精性肝损伤模型的药物保肝活性。微流控芯片运行正常后，向第二三维培养通道2c内加入浓度为425μM的乙醛48h，更换流动培养通道2a中培养液为包含浓度为0、240、480、960μg/mL氧化苦参碱的市售肝细胞完全培养基，每24h检测第二三维培养通道2c上清液中尿素氮含量，流动培养通道2a上清液中白蛋白含量，每48h检测芯片流动培养通道2a、第一三维培养通道2b、第二三维培养通道2c上清液中一型前胶原和基质金属蛋白酶抑制剂含量，以此评价氧化苦参碱对乙醛诱导的酒精性肝损伤的治疗作用。
实施例6
本实施例采用实施例1中的微流控芯片模型，利用该微流控芯片评价基于脂多糖诱导的肝纤维化模型的抗肝纤维化药物活性。微流控芯片运行正常后，流动培养通道2a内循环含1μg/mL脂多糖的市售肝细胞完全培养基，循环72h后，更换流动培养通道2a中培养液为包含浓度为0、240、480、960μg/mL氧化苦参碱的市售肝细胞完全培养基，每24h检测第二三维培养通道2c上清液中尿素氮含量，流动培养通道2a上清液中白蛋白含量，每48h检测芯片流动培养通道2a、第一三维培养通道2b、第二三维培养通道2c上清液中一型前胶原和基质金属蛋白酶抑制剂含量，以此评价氧化苦参碱对脂多糖诱导的肝纤维化的抑制作用。
实施例7
本实施例采用实施例1中的微流控芯片模型，利用该微流控芯片评价基于白介素6(IL-6)、转化生长因子β(TGF-β)和肿瘤坏死因子α(TNF-α)三种炎症配体激活终产物诱导的中后期肝纤维化模型的抗肝纤维化药物活性。微流控芯片运行正常后，向流动培养通道2a循环包含浓度为100pg/mL TNF-α的市售肝细胞完全培养基，向第一三维培养通道2b加入包含浓度为5ng/mL TGF-β的无血清市售肝细胞培养基，向第二三维培养通道2c加入包含浓度为50pg/mL IL-6的无血清市售肝细胞培养基，循环72h后，更换流动培养通道2a中培养液为包含浓度为0、240、480、960μg/mL氧化苦参碱的市售肝细胞完全培养基，每24h检测第二三维培养通道2c上清液中尿素氮含量，流动培养通道2a上清液中白蛋白含量，每48h检测芯片流动培养通道2a、第一三维培养通道2b、第二三维培养通道2c上清液中一型前胶原和基质金属蛋白酶抑制剂含量，以此评价氧化苦参碱对基于多种炎症配体激活终产物诱导的中晚期肝纤维化损伤的抑制作用。

Claims (8)

1.一种平行通道微栅栏共培养多种细胞的微流控芯片，其特征在于，所述的微流控芯片包括一层基板和一层基片，所述的基板上包括流动培养通道和三维培养通道；所述的流动培养通道的两端设置储液池，三维培养通道的两端设置三维培养通道进出液口；流动培养通道、与流动培养通道邻近的三维培养通道之间设置模拟内皮屏障的微栅栏结构，不同的三维培养通道之间设置三维培养通道间微栅栏，作为物质交换和细胞迁移的通道；所述的模拟内皮屏障的微栅栏结构与三维培养通道间微栅栏仅布置于流动培养通道和三维培养通道的底端。
2.根据权利要求1所述的一种平行通道微栅栏共培养多种细胞的微流控芯片，其特征在于，所述的模拟内皮屏障的微栅栏结构与三维培养通道间微栅栏的设置高度为流动培养通道和三维培养通道高度的1/40。
3.根据权利要求1所述的一种平行通道微栅栏共培养多种细胞的微流控芯片，其特征在于，所述基片的材料选自石英、玻璃、PMMA或聚碳酸酯中的一种。
4.根据权利要求1所述的一种平行通道微栅栏共培养多种细胞的微流控芯片，其特征在于，所述基板的材料选自石英、玻璃、PMMA、PDMS聚合物、聚酯、硅胶、琼脂糖、壳聚糖或海藻酸钠中的一种。
5.权利要求1-4任一所述的一种平行通道微栅栏共培养多种细胞的微流控芯片的应用，其特征在于，应用于模拟肝血窦内多种肝细胞空间排布方面，应用于模拟血脑屏障结构多种神经细胞空间排布方面，应用于模拟肠道屏障功能多种肠道细胞空间排布方面，应用于肾单位结构中多种肾脏细胞空间排布方面。
6.根据权利要求5所述的应用，其特征在于，当所述微流控芯片应用于模拟肝血窦内多种肝细胞空间排布方面时，所述流动培养通道和三维培养通道内通入的细胞根据需求，选自人、任何一种动物的肝窦内皮细胞、肝星状细胞、肝实质细胞和枯否细胞衍生的细胞系，或选自来源于人、任何一种动物的肝相关原代细胞，或使用iPS诱导分化的肝相关功能细胞。
7.根据权利要求6所述的应用，其特征在于，所述微流控芯片设置一条流动培养通道和两条三维培养通道；所述流动培养通道中接种肝血窦内皮细胞，与流动培养通道邻近的三维培养通道进液口通入混有肝星状细胞的三维胶，另一条三维培养通道进液口通入混有肝实质细胞的三维胶，所述流动培养通道通入枯否细胞静置后，脱色摇床无泵驱动循环市售肝细胞生长完全培养基，两条三维培养通道灌满无血清的市售肝细胞完全培养基。
8.根据权利要求6所述的应用，其特征在于，所述三维胶选自BME、Matrixgel或海藻酸钠胶中的一种。
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