CN115340948A - 一种逐层叠加共培养多种肝细胞的微流控芯片及其应用 - Google Patents

一种逐层叠加共培养多种肝细胞的微流控芯片及其应用 Download PDF

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CN115340948A
CN115340948A CN202211010404.XA CN202211010404A CN115340948A CN 115340948 A CN115340948 A CN 115340948A CN 202211010404 A CN202211010404 A CN 202211010404A CN 115340948 A CN115340948 A CN 115340948A
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hepatocytes
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魏文博
陈宗正
高志刚
张秀莉
罗勇
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Shenzhen Second Peoples Hospital
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，一种逐层叠加共培养多种肝细胞的微流控芯片及其应用，其中微流控芯片分为底层的流体通道、中间层三维培养区和储液池以及上层盖板三部分组成。芯片整体包含多个培养单元，底层流体通道连接中间层两侧储液池，将细胞培养区域串联，形成单侧流动。中间层的体三维培养区与底层的圆形培养区域对应，中间由两层多孔膜隔开，上层盖板将细胞培养区域和储液池上口覆盖。本发明将多种肝细胞分别接种在两层多孔膜和三维培养区内，在一块微流控芯片上形成多个串联的肝血窦模型单元，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

一种逐层叠加共培养多种肝细胞的微流控芯片及其应用
技术领域
本发明涉及一种逐层叠加共培养多种肝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
微流控技术是研究微/亚微米级流体控制的技术。微流控芯片属于其中的一个分支。最初，微流控芯片被用来取代传统的实验室分析方法，通过显微层析和毛细管电泳构建“μ-Tas”或“芯片上的实验室”分析模型。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)
肝脏具有许多特殊的生理功能，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。肝脏主要由肝实质细胞组成，同时含有多种非实质细胞(肝窦内皮细胞、肝星状细胞和枯否细胞)。几种主要的肝细胞组成肝脏的基本解剖学单元以完成生命活动中各种生化反应，包括合成和分解生命活动必须的小分子或大分子。
肝血窦是肝脏中不同细胞类型组成的微血管单位，内嵌有肝窦内皮细胞，内皮上散布肝内原位巨噬细胞枯否细胞。肝星状细胞主要存在于窦周间隙中，窦周间隙是肝细胞与血窦内皮细胞之间的狭窄间隙，其中充满来自血窦的血浆，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。肝细胞沿肝窦呈放射状排列，组成肝脏功能的基本功能单元肝小叶，以支持肝脏功能。
基于器官芯片技术对多种肝细胞共培养和流动培养可以大幅提高体外肝脏模型的生理特性，提升肝脏基本功能。格里菲斯等人(Functional behavior of primary ratliver cells in a three-dimensional perfused microarray bioreactor.Tissueengineering.2002)构建了一个结合微流控流动培养和多个细胞类型的复杂细胞组织。使用原代大鼠肝细胞和非实质细胞以一定比例(19：1)进行球状培养并接种在微加工的PDMS芯片腔室中。扫描电子显微镜显示生物反应器中形成的肝组织中具有稳定的糖原储存，且包含胆总管以及血管状结构。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。杜等人(Mimicking liver sinusoidal structures and functionsusing a 3D-configured microfluidic chip.Lab on a Chip.2017)构建了一种逐层叠加式的肝血窦芯片模型。利用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。但现存的这类技术使用的细胞种类较少，培养方式单一且存在低通量、稳定性差和对大型泵阀设备依赖的缺陷。
发明内容
基于逐层叠加(layer-by-layer)这一经典结构，同时克服细胞种类少、操作繁复、连接大型设备和低通量的技术不足，本发明目的是提供一种逐层叠加共培养多种肝细胞的微流控芯片及其应用。该微流控芯片利用无泵驱动的流体流动方式，设置三个串联的肝血窦单元，能够较高通量地模拟肝脏内细胞的空间排布，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：
一种逐层叠加共培养多种肝细胞的微流控芯片，包括底层基板、中间层基板和上层盖板三层结构；其中，底层基板上设有多个串联的细胞培养区，两端的细胞培养区与储液区连通，共同形成底层流体通道；中间层基板上设有多个单独的三维培养区和储液池，分别与底层基板上的细胞培养区与储液区一一对应；三维培养区与细胞培养区间由两层多孔膜隔开；底层基板和中间层基板上对称设有多个安装孔，通过铆钉将二者固定为一体，上层盖板盖在中间层基板上。
使用时，不同的非实质肝细胞分别接种在多孔膜两侧，细胞培养区和三维培养区内加入混有实质肝细胞的三维胶；细胞接种完成后，底层通道及储液池内灌注模拟血液的培养基，三维培养区内灌注无血清肝细胞完全培养基，脱色摇床无泵驱动底层通道内液体循环流动。
所述的底层基板、中间层基板和上层盖板的材料选自石英、玻璃、PMMA、PDMS聚合物中的一种或两种以上，所述的多孔膜选自聚碳酸酯、聚酯、琼脂糖、壳聚糖或海藻酸钠中的一种或两种以上，所述的接种在多孔膜上的非实质肝细胞及三维培养的肝实质细胞根据需求，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、原代细胞或iPS诱导分化的功能细胞的一种或两种以上。
所述的细胞培养区的内表面进行疏水防渗漏处理。
所述的底层通道宽0.9mm，深度2mm，两侧连接的储液区尺寸为5mm*5mm，细胞培养区有三个，为直径为6mm的圆形；所述的三维培养区为底面直径6mm、深5mm的圆柱体结构，储液池为边长5mm的立方体。
所述的微流控芯片可应用于模拟肝血窦内多种肝细胞空间排布、模拟肝空间结构、建立肝疾病模型、药物研发。
本发明的有益效果是：本发明的微流控芯片是一种较大通量、结构简单、稳定性高并且可多次使用的逐层叠加式肝血窦芯片模型。结合二维、三维培养方式，采用无泵驱动的半开放式单侧流动运行，提供了流体剪切力及近生理的细胞排布和微环境。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是本发明一种逐层叠加共培养4种肝细胞的微流控芯片结构示意图。
图中：1、底层基板，1a、细胞培养区，1b储液区，1c、第一层多孔膜，1d、第二层多孔膜，2、中间层基板，2a、三维培养区，2b、储液池，3、上层盖板。
图2(a)和图2(b)是实施例1中肝血窦芯片基本肝功能测试的结果图，其中，图2(a)是蛋白质合成的结果，图2(b)是尿素合成的结果。
图3是实施例2中不同浓度乙醛诱导的酒精性肝损伤检测的结果图。
图4(a)和图4(b)是实施例3中乙醛诱导的肝纤维化检测的结果图，其中，图4(a)是蛋白质合成的结果，图4(b)是尿素合成的结果。
具体实施方式
以下结合附图和技术方案，进一步说明本发明的具体实施方式。
本发明的一种逐层叠加共培养4种肝细胞的微流控芯片如图1所示，包括底层基板1、中间层基板2和上层盖板3。其中，底层基板1上设有多个串联的细胞培养区1a，两端的细胞培养区1a与储液区1b连通，共同形成底层通道；中间层基板2上设有分别与底层基板1上的细胞培养区1a与储液区1b相对应的三维培养区2a和储液池2b；三维培养区2a与细胞培养区1a间由第一层多孔膜1c和第二层多孔膜1d隔开；底层基板1和中间层基板2通过铆钉连接为一体，上层盖板盖3在中间层基板2上。
实施例1
本实施例中，底层基板1、中间层基板2和上层盖板3均为PMMA加工，且底层基板1表面作疏水防渗漏处理。底层基板1上设有三个直径6mm的串联的圆形细胞培养区1a，两侧为边长5mm正方形的储液区1b。中间层基板2上设有三个的直径6mm三维培养区2a，两侧为边长为5mm储液池2b，三维培养区2a与细胞培养区2a对应，储液池2b与储液区1b对应。上层盖板3覆盖三维培养区2a和储液池2b上；第一层多孔膜1c和第二层多孔膜1d为聚碳酸酯多孔膜。
第一层多孔膜1c下表面接种HUVEC细胞和附着在HUVEC细胞表面的U937细胞，第二层多孔膜1d上表面接种LX-2细胞，所述三维培养区2a内加入混有Matrixgel的HepG2细胞，三维培养区2a混有Matrixgel的HepG2细胞静置后，上方灌注无血清的市售HepG2细胞生长完全培养基，储液池2b中通入模拟血液完全培养基，脱色摇床驱动循环。对所述逐层叠加共培养4种肝细胞的微流控芯片液体进行基本肝功能测试，结果如图2(a)和图2(b)所示，随着时间增加，芯片上肝功能维持良好，并显著高于孔板中的体外肝脏模型。
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，三维培养区内更换为浓度为0、425、850、1700μM的乙醛的市售无血清HepG2细胞生长完全培养基48h后，检测三维培养区上清液中白蛋白含量，表征微流控芯片体外肝模型的代谢功能，结果如图3所示，随乙醛浓度升高，白蛋白分泌量降低，体外肝脏模型合成蛋白功能受损。
实施例3
本实施例采用实施例1中的微流控芯片模型，以乙醛诱导的肝纤维化检测为例，微流控芯片运行正常以后，三维培养区内更换为包含浓度为425μM的乙醛的市售无血清HepG2细胞生长完全培养基，每24h取出三维培养区和底层通道内液体进行白蛋白和尿素氮含量检测，观察微流控芯片内肝模型的基本肝功能变化，结果如图4(a)和图4(b)所示，随着作用时间的延长，微流控芯片肝模型的白蛋白代谢功能明显下降，尿素排泄功能也受影响。
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脂多糖诱导的肝纤维化模型。微流控芯片运行正常后，向底层流体通道循环包含浓度为1μg/mL的脂多糖的模拟血液培养基，每24h取三维培养区和底层通道内液体进行白蛋白和尿素氮含量检测，观察微流控芯片内肝模型的基本肝功能变化。
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肝损伤模型的保肝药物活性。微流控芯片运行正常后，三维培养区内更换为包含浓度为425μM的乙醛的市售无血清HepG2细胞生长完全培养基48h后，更换底层流体通道中培养液为包含浓度为0、240、480、960μg/mL氧化苦参碱的模拟血液培养基，每24h取三维培养区和底层通道内液体进行白蛋白和尿素氮含量检测，继续循环72h后取出三维培养区和底层通道内液体进行一型前胶原和基质金属蛋白酶抑制剂的含量检测，以此评价氧化苦参碱对乙醛诱导的肝损伤的治疗作用。
实施例6
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，向底层流体通道循环包含浓度为1μg/mL脂多糖的模拟血液培养基，循环72h后，更换底层流体通道中培养液为包含浓度为0、240、480、960μg/mL氧化苦参碱的模拟血液培养基，每24h取三维培养区和底层通道内液体进行白蛋白和尿素氮含量检测，继续循环72h后取出三维培养区和底层通道内液体进行一型前胶原和基质金属蛋白酶抑制剂的含量检测，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。

Claims (9)

1.一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的微流控芯片包括底层基板、中间层基板和上层盖板三层结构；其中，底层基板上设有多个串联的细胞培养区，两端的细胞培养区与储液区连通，共同形成底层流体通道；中间层基板上设有多个单独的三维培养区和储液池，分别与底层基板上的细胞培养区与储液区一一对应；三维培养区与细胞培养区间由两层多孔膜隔开；底层基板和中间层基板上对称设有多个安装孔，通过铆钉将二者固定为一体，上层盖板盖在中间层基板上。
2.根据权利要求1所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，使用时，不同的非实质肝细胞分别接种在多孔膜两侧，细胞培养区和三维培养区内加入混有实质肝细胞的三维胶；细胞接种完成后，底层通道及储液池内灌注模拟血液的培养基，三维培养区内灌注无血清肝细胞完全培养基，脱色摇床无泵驱动底层通道内液体循环流动。
3.根据权利要求1或2所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的底层基板、中间层基板和上层盖板的材料选自石英、玻璃、PMMA、PDMS聚合物中的一种或两种以上，所述的多孔膜选自聚碳酸酯、聚酯、琼脂糖、壳聚糖或海藻酸钠中的一种或两种以上，所述的接种在多孔膜上的非实质肝细胞及三维培养的肝实质细胞根据需求，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、原代细胞或iPS诱导分化的功能细胞的一种或两种以上。
4.根据权利要求1或2所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的细胞培养区的内表面进行疏水防渗漏处理。
5.根据权利要求3所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的细胞培养区的内表面进行疏水防渗漏处理。
6.根据权利要求1或2或5所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的底层通道宽0.9mm，深度2mm，两侧连接的储液区尺寸为5mm*5mm，细胞培养区有三个，为直径为6mm的圆形；所述的三维培养区为底面直径6mm、深5mm的圆柱体结构，储液池为边长5mm的立方体。
7.根据权利要求3所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的底层通道宽0.9mm，深度2mm，两侧连接的储液区尺寸为5mm*5mm，细胞培养区有三个，为直径为6mm的圆形；所述的三维培养区为底面直径6mm、深5mm的圆柱体结构，储液池为边长5mm的立方体。
8.根据权利要求4所述的一种逐层叠加共培养多种肝细胞的微流控芯片，其特征在于，所述的底层通道宽0.9mm，深度2mm，两侧连接的储液区尺寸为5mm*5mm，细胞培养区有三个，为直径为6mm的圆形；所述的三维培养区为底面直径6mm、深5mm的圆柱体结构，储液池为边长5mm的立方体。
9.权利要求1-8任意所述的一种逐层叠加共培养多种肝细胞的微流控芯片应用于模拟肝血窦内多种肝细胞空间排布、模拟肝空间结构、建立肝疾病模型、药物研发。
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