CN111269833A - 一种基于器官芯片的人胰岛类器官模型构建方法 - Google Patents
一种基于器官芯片的人胰岛类器官模型构建方法 Download PDFInfo
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Abstract
本发明公开了一种基于器官芯片的人胰岛类器官模型构建方法,特别是利用人诱导多能干细胞原位生成的拟胚体诱导发育成为含有多细胞成分的胰岛类器官,该器官芯片为从可灌注多层荚膜三维组织培养芯片,通过灌注含有不同小分子诱导剂的分化培养基促进拟胚体向胰岛类组织分化。同时芯片上下层通道内同时灌注诱导分化培养基,灌注速率为100μL h‑1,基于此多层芯片流体灌注培养体系诱导分化形成的人源胰岛组织含有胰岛β细胞和胰岛α细胞,类胰组织细胞有良好的细胞活力并且可实现体外长期培养至60天,具有良好的胰岛素分泌功能,实现体外构建仿生人胰岛组织的用于胰岛组织发育研究和胰岛病理生理机制的研究,为相关药物评价及筛选提供有利平台。
Description
技术领域
本发明涉及将微流控芯片技术应用到体外构建人源器官模型的技术领域,具体涉及一种基于器官芯片的人胰岛类器官模型构建方法。
背景技术
近年来糖尿病病发率急剧上升,已成为世界第三大慢性病严重威胁社会健康。糖尿病根据其发病机制不同可分为由胰岛素不足引起的1型糖尿病和由胰岛素抵抗引起的2型糖尿病。1型糖尿病是由自身免疫系统破坏机体胰腺组织β细胞,胰岛素分泌不足从而引发机体血糖升高。目前胰腺或胰岛移植是从根本上治疗1型糖尿病的主要方法,但由于供者数量缺乏,移植后免疫排斥反应等问题,限制该方法在临床治疗中的大规模应用。寻找一种功能、数量都与正常β细胞相匹配的替代细胞是亟待解决的问题。诱导型多能干细胞不仅能够体外诱导生成正常的心肌、神经、肝脏等细胞,还能够以患者体细胞为来源体细胞生成特异性的细胞类型。最新研究表明,以IPS细胞为基础发展出来的类器官模型,能够模拟出器官的发育形成过程,可定向向三胚层发育,含有多种细胞成分。这些都为疾病治疗、药物筛选和再生性医学提供新方法。IPS诱导胰岛技术因其细胞来源广,自体移植免疫排斥低等优点,是目前胰岛移植理想细胞来源,成为研究细胞治疗1型糖尿病的新热点。
器官芯片技术飞速发展,因其能够多空间角度结合三维细胞基质,流体剪切力,氧浓度梯度等生物物理化学因素,已经在芯片上构建出功能良好的肺,心肌,肝等微器官模型。虽然,有一些报道应用微流控技术研究IPS诱导胰岛,此方法一定程度上促进胰岛诱导效率,但并未解决胰岛移植的根本问题。如何借助器官芯片技术建立体外功能良好的胰岛模型,大规模,稳定的胰岛细胞用于在糖尿病的治疗和药物测评,基础研究将是本次发明的重点。
发明内容
针对现有技术存在的问题,本发明的目的在于提供一种生物相容性和透光性良好的器官芯片以及基于器官芯片的人胰岛类器官模型构建方法,可以大规模,稳定的将人干细胞来源的胰岛细胞用于在糖尿病的治疗和药物测评中。
一种器官芯片,基于微流控芯片技术,该芯片主要由4层结构粘合封接而成,自上而下依次包括PDMS上层1,PDMS贯穿孔层2,聚碳酸酯膜层3,PDMS底层4;其中,所述PDMS上层1与PDMS贯穿孔层2封接相连;PDMS贯穿孔层2与聚碳酸酯膜层3通过PDMS胶体粘合连接,聚碳酸酯膜层3与PDMS底层4封接相连;
所述PDMS上层1包含细胞入口池5,上层培养基灌流室6、废液池7和上层灌流通道14;所述上层培养基灌流室6上端与细胞入口池5相连,下端与废液池7相连;
所述PDMS贯穿孔层2上设有细胞培养室8,PDMS贯穿孔层2上的贯穿孔与聚碳酸酯膜层3形成细胞培养小室13,细胞培养室8底部设有细胞培养室底9;所述细胞培养室8与细胞培养室底9通过PDMS粘合;
所述PDMS底层4包括下层培养基入口池10,下层培养基灌流室11、下层废液池12和下层灌流通道15;所述下层培养基灌流室11上端与下层培养基入口池10相连,下端与下层废液池12相连;
所述PDMS贯穿孔层2和聚碳酸酯膜层3多孔结构,实现上下层营养物和代谢产物的物质交换。
所述细胞培养小室13的个数为100~200个。细胞培养小室13可进行编码,对单个干细胞来源的拟胚体进行原位诱导,并对发育过程,病理生理改变进行原位追踪观察。
所述器官芯片上层灌流通道14和下层灌流通道15高度为300-500μm;所述细胞培养小室13半径为200-300μm,高度为250-400μm;PDMS贯穿孔膜2厚度为300μm,贯穿孔直径500μm;聚碳酸脂膜3厚度4-8nm,孔径4nm。
本发明提供的器官芯片,细胞培养小室13底部结构为多孔膜结构,细胞培养小室13上层培养基灌流通道14和下层培养基灌流通道15可同时添加流体,可以实现上下层营养物和代谢产物的物质交换。
一种基于器官芯片的多能干细胞来源的人胰岛类器官构建方法,采用了上述器官芯片,具体步骤如下:
(1)将hiPSCs细胞悬液由细胞入口池5注入芯片上,静置10min将培养室外散在细胞冲出,过夜培养后形成拟胚体,灌注诱导培养基,开始胰岛类器官诱导;
(2)人源诱导多能干细胞形成的拟胚体向胰岛类器官待诱导过程结束后,进行细胞死活染色实验验证细胞活性,蛋白及基因水平上鉴定胰岛的细胞种类,胰岛β细胞的成熟度,胰岛的内分泌功能,胰岛内分泌量及分泌时效性检测用以检测胰岛分泌功能。
所述细胞为人来源的多能干细胞。所述人来源的多能干细胞为人胚胎干细胞hESCs、人诱导多能干细胞hiPSCs。
所述步骤(2)检测胰岛分泌功能之前需要用含20-30mM葡萄糖的KRBH缓冲液处理。
所述诱导培养基具体如下:
第一阶段内胚层诱导:DMEM/F12基础培养基加0.2%BSA,50ng/ml activin A,3μMCHIR99021,2mM LiCl处理一天;之后用DMEM/F12基础培养基加0.2%BSA,1%B27,和50ng/ml activin A处理4天;
第二阶段胰向内胚层诱导:DMEM基础培养基加0.5%B27,2μM retinoic acid,2μMdorsomorphin,10μM SB431542,5ng/ml bFGF和250nm SANT-1,维持6天;
第三阶段向内分泌前体细胞诱导:DMEM基础培养基加0.5%B27s,50μg/mlascorbic acid,2μM dorsomorphin,10μM SB431542和10μM DAPT,维持4天;
第四阶段向内分泌细胞诱导:CMRL 1066基础培养基加0.5%B27supplement,0.5%penicillin–streptomycin,25mM glucose,2μM dorsomorphin,10μM SB431542,10mMnicotinamide和50μg/ml ascorbic acid,维持8天。
本发明提供了一种基于微流控芯片技术的可高通量获得一种干细胞来源的人胰岛类器官模型。
本发明建立的体外干细胞来源的人胰岛类器官模型,含有两种主要胰岛组织细胞成分胰岛β细胞和胰岛α细胞。
本发明建立的体外干细胞来源的人胰岛类器官模型,具有良好的胰岛素分泌功能,模拟体内生理条件下胰岛分泌胰岛素的糖刺激响应过程。
本发明建立的体外干细胞来源的人胰岛类器官模型,类胰组织细胞有良好的细胞活力并且可实现体外长期培养至60天。
本发明提供了一种基于微流控芯片技术的体外构建干细胞来源的人胰岛类器官的构建方法,所用起始细胞类型为人诱导多能干细胞(hiPSCs)。
1)基于微流控芯片的人胰岛类器官芯片的构建
利用器官芯片技术,多维结合三维细胞培养方式,流体剪切力,构建出功能hiPSc来源的胰岛类器官,所构建的类组织细胞成熟具有良好的胰岛素分泌功能,多细胞组分(胰岛β细胞、胰岛α细胞),有效的糖刺激反应(GSIS反应)。阶梯式诱导的方法来模拟体内胰腺发育的过程,使多能性干细胞逐步经过内胚层细胞、胰岛前体细胞等阶段,最后发育为成熟胰岛β细胞。
2)芯片上人胰岛类器官的功能评价
构建结构和功能与生理环境更加接近的胰岛类器官是发明目的关键内容。利用多层三维灌注培养芯片系统,结合hiPSc细胞诱导技术原位形成并分化成胰岛类器官。结合物理微环境---流体因素对胰岛发育,胰岛成熟及胰岛长期体外模型构建的影响并探讨可能的影响机制。鉴定胰岛相关成熟蛋白和基因的表达,胰岛素分泌的糖刺激反应。该工程化的体外胰岛器官芯片的构建,可成潜在的为优化胰岛来源方式用于糖尿病胰岛移植治疗及相关糖尿病药物评价提供有利平台。
本发明提供的基于微流控器官芯片技术的人胰岛类器官模型,可采用生物学上常用的细胞检测手段对迁移运动到胶原中的细胞进行检测,包括细胞死活标记染色、细胞免疫荧光染色、PCR检测、蛋白质检测。
本发明优点:
本发明利用微流控技术,以具有良好生物相容性,透光性的PDMS为芯片材料,设计的装置可横向直接记录和观察细胞迁移行为的芯片,功能完备,操作简单,并且在芯片上可以独立完成各项信号检测,如细胞蛋白表达,细胞因子分泌,细胞增殖,凋亡检测等。
附图说明
下面结合附图及实施方式对本发明作进一步详细的说明:
图1:本发明器官芯片整体结构示意图;
图2:芯片上hiPSCs来源的胰岛类器官发育过程明场追踪图;
图3:芯片上hiPSCs来源的胰岛类器官细胞活性检测及长时间培养活性检测;
图4:芯片上hiPSCs来源的胰岛类器官细胞成分鉴定;
图5:芯片上hiPSCs来源的胰岛类器官胰岛素分泌功能检测。
其中,PDMS上层1,PDMS贯穿孔层2,聚碳酸酯膜层3,PDMS底层4,细胞入口池5,上层培养基灌流室6,废液池7,细胞培养室8,细胞培养室底9,下层培养基入口池10,下层培养基灌流室11,下层废液池12,胞培养小室13,上层灌流通道14,下层灌流通道15。
具体实施方式
下面结合具体实施例对本发明方法作详细说明,本实施例在以本发明技术方案为前提下进行实施,但本发明的保护范围不限于下述的实施例。
实施例1
芯片上构建诱导多能干细胞来源的人胰岛类器官及相关胰岛内分泌功能的评价。
利用实验室自行设计并制作的器官芯片,构型如图1所示。器官芯片主要基于微流控芯片,由4层结构粘合封接而成,包括PDMS上层1,PDMS贯穿孔层2,聚碳酸酯膜层3,PDMS底层4,其中包含细胞入口池5,上层培养基灌流室6,废液池7,细胞培养室8,细胞培养室底9,下层培养基入口池10,下层培养基灌流室11,下层废液池12。其中PDMS上层1与PDMS贯穿孔层2封接相连;PDMS贯穿孔层2与聚碳酸酯膜层3相粘连,聚碳酸酯膜层3与PDMS底层4封接相连;上层培养基灌流室6上连细胞入口池5,上层培养基灌流室6下连废液池7;细胞培养室8与细胞培养室底9相连接,每个贯穿孔可与聚碳酸酯膜形成细胞培养小室,装置中含6×5个整列细胞球培养小室;下层培养基灌流室11上连下层培养基入口池10,下层培养基灌流室11下连下层废液池12。
将hiPSCs细胞悬液由细胞入口池5注入芯片上,静置10min将培养室外散在细胞冲出,过夜培养后形成拟胚体,灌注诱导培养基开始胰岛类器官诱导(图2)。待诱导过程结束后,继续原位灌注培养形成胰岛类器官,实现胰岛组织体外的长期培养,经细胞live、dead染色实验结果显示,所形成的胰岛类组织可维持良好细胞活性长达60天(图3)。在灌注培养体系下发育形成的胰岛类器官,从蛋白及基因水平上鉴定胰岛的细胞种类(INS,CGC),胰岛β细胞的成熟度(PDX1,NKX6.1),胰岛的内分泌功能(INS,GCG)。胰岛的内分泌功能主要体现为胰岛素的分泌量及分泌时效性,为测量所诱导胰岛类组织的胰岛素分泌功能,首先用KRBH(含2.5mM葡萄糖)缓冲液培养箱内培养1h,后用含有2.5mM和25mM缓冲液1ml交替作用在胰岛类组织(100-150微球),30min后收集上清,进行ELISA检测,结果如图5所示,芯片上所形成的胰岛类组织有良好的胰岛素分泌功能和灵敏的糖刺激反应。
Claims (10)
1.一种器官芯片,其特征在于:该芯片主要由4层结构粘合封接而成,自上而下依次包括PDMS上层(1),PDMS贯穿孔层(2),聚碳酸酯膜层(3),PDMS底层(4);其中,所述PDMS上层(1)与PDMS贯穿孔层(2)封接相连;PDMS贯穿孔层(2)与聚碳酸酯膜层(3)通过PDMS胶体粘合连接,聚碳酸酯膜层(3)与PDMS底层(4)封接相连;
所述PDMS上层(1)包含细胞入口池(5),上层培养基灌流室(6)、废液池(7)和上层灌流通道(14);所述上层培养基灌流室(6)上端与细胞入口池(5)相连,下端与废液池(7)相连;
所述PDMS贯穿孔层(2)上设有细胞培养室(8),PDMS贯穿孔层(2)上的贯穿孔与聚碳酸酯膜层(3)形成细胞培养小室(13),细胞培养室(8)底部设有细胞培养室底(9);所述细胞培养室(8)与细胞培养室底(9)通过PDMS粘合;
所述PDMS底层(4)包括下层培养基入口池(10),下层培养基灌流室(11)、下层废液池(12)和下层灌流通道(15);所述下层培养基灌流室(11)上端与下层培养基入口池(10)相连,下端与下层废液池(12)相连;
所述PDMS贯穿孔层(2)和聚碳酸酯膜层(3)多孔结构,实现上下层营养物和代谢产物的物质交换。
2.根据权利要求1所述的器官芯片,其特征在于:所述细胞培养小室(13)的个数为100~200个。
3.根据权利要求1所述的器官芯片,其特征在于:所述器官芯片上层灌流通道(14)和下层灌流通道(15)高度为300-500μm;所述细胞培养小室(13)半径为200-300μm,高度为250-400μm;PDMS贯穿孔膜(2)厚度为300μm,贯穿孔直径500μm;聚碳酸脂膜(3)厚度4-8nm,孔径4nm。
4.根据权利要求1所述的多器官芯片,其特征在于:所述细胞培养小室(13)可进行编码,对单个干细胞来源的拟胚体进行原位诱导,并对发育过程,病理生理改变进行原位追踪观察。
5.根据权利要求1所述的多器官芯片,其特征在于:所述上层灌流通道(14)和下层灌流通道(15)可同时添加流体,实现良好的物质交换。
6.一种基于器官芯片的人胰岛类器官模型构建方法,其特征在于采用了权利要求1和2中任一种的芯片,具体步骤如下:
(1)将hiPSCs细胞悬液由细胞入口池(5)注入芯片上,静置10min将培养室外散在细胞冲出,过夜培养后形成拟胚体,灌注诱导培养基,开始胰岛类器官诱导;
(2)人源诱导多能干细胞形成的拟胚体向胰岛类器官待诱导过程结束后,进行细胞死活染色实验验证细胞活性,蛋白及基因水平上鉴定胰岛的细胞种类,胰岛β细胞的成熟度,胰岛的内分泌功能,胰岛内分泌量及分泌时效性检测用以检测胰岛分泌功能。
7.根据权利要求6所述的基于器官芯片的人胰岛类器官模型构建方法,其特征在于:所述细胞为人来源的多能干细胞。
8.根据权利要求7所述的基于器官芯片的人胰岛类器官模型构建方法,其特征在于:所述人来源的多能干细胞为人胚胎干细胞hESCs、人诱导多能干细胞hiPSCs。
9.根据权利要求6所述的基于器官芯片的人胰岛类器官模型构建方法,其特征在于:所述步骤(2)检测胰岛分泌功能之前需要用含20-30mM葡萄糖的KRBH缓冲液处理。
10.根据权利要求6所述的基于器官芯片的人胰岛类器官模型构建方法,其特征在于:所述诱导培养基具体如下:
第一阶段内胚层诱导:DMEM/F12基础培养基加0.2%BSA,50ng/ml activin A,3μMCHIR99021,2mM LiCl处理一天;之后用DMEM/F12基础培养基加0.2%BSA,1%B27,和50ng/ml activin A处理4天;
第二阶段胰向内胚层诱导:DMEM基础培养基加0.5%B27,2μM retinoic acid,2μMdorsomorphin,10μM SB431542,5ng/ml bFGF和250nm SANT-1,维持6天;
第三阶段向内分泌前体细胞诱导:DMEM基础培养基加0.5%B27s,50μg/ml ascorbicacid,2μM dorsomorphin,10μM SB431542和10μM DAPT,维持4天;
第四阶段向内分泌细胞诱导:CMRL 1066基础培养基加0.5%B27 supplement,0.5%penicillin–streptomycin,25mM glucose,2μM dorsomorphin,10μM SB431542,10mMnicotinamide和50μg/ml ascorbic acid,维持8天。
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CN112553076A (zh) * | 2020-12-15 | 2021-03-26 | 东南大学 | 脑类器官体外培养芯片 |
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Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070231887A1 (en) * | 2006-03-14 | 2007-10-04 | University Of Rochester | Cell culture devices having ultrathin porous membrane and uses thereof |
WO2008066199A1 (fr) * | 2006-12-01 | 2008-06-05 | Naoya Kobayashi | Procédé d'induction de la différenciation de cellules souches embryonnaires en cellules sécrétrices d'insuline, cellules sécrétrices d'insuline induites par le procédé et leur utilisation |
WO2012032646A1 (ja) * | 2010-09-10 | 2012-03-15 | 株式会社島津製作所 | 細胞培養デバイス及び細胞培養方法 |
TW201439537A (zh) * | 2012-10-25 | 2014-10-16 | Academia Sinica | 用於氧氣濃度控制下同步細胞培養的微流體陣列平台 |
WO2015088072A1 (ko) * | 2013-12-11 | 2015-06-18 | 한국과학기술원 | 인간 다능성 줄기세포로부터 인슐린 생산 배타세포의 내분비 응집체의 제조방법 |
CN106811413A (zh) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | 基于微流控技术的多器官芯片及其制备方法 |
US20170248583A1 (en) * | 2014-10-27 | 2017-08-31 | The Governing Council Of The University Of Toronto | Microfluidic device for cell-based assays |
WO2017175236A1 (en) * | 2016-04-06 | 2017-10-12 | Dandekar Jain Prajakta | Microfluidic platform for developing in-vitro co-cultures of mammalian tissues. |
CN107502547A (zh) * | 2017-09-25 | 2017-12-22 | 中科芯瑞(苏州)生物科技有限公司 | 一种实现多种细胞共培养的微流控芯片及其应用 |
CN108641931A (zh) * | 2018-04-04 | 2018-10-12 | 浙江大学 | 一种数字化微阵列器官芯片及其应用 |
-
2018
- 2018-12-05 CN CN201811477503.2A patent/CN111269833A/zh active Pending
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070231887A1 (en) * | 2006-03-14 | 2007-10-04 | University Of Rochester | Cell culture devices having ultrathin porous membrane and uses thereof |
WO2008066199A1 (fr) * | 2006-12-01 | 2008-06-05 | Naoya Kobayashi | Procédé d'induction de la différenciation de cellules souches embryonnaires en cellules sécrétrices d'insuline, cellules sécrétrices d'insuline induites par le procédé et leur utilisation |
WO2012032646A1 (ja) * | 2010-09-10 | 2012-03-15 | 株式会社島津製作所 | 細胞培養デバイス及び細胞培養方法 |
TW201439537A (zh) * | 2012-10-25 | 2014-10-16 | Academia Sinica | 用於氧氣濃度控制下同步細胞培養的微流體陣列平台 |
WO2015088072A1 (ko) * | 2013-12-11 | 2015-06-18 | 한국과학기술원 | 인간 다능성 줄기세포로부터 인슐린 생산 배타세포의 내분비 응집체의 제조방법 |
US20170248583A1 (en) * | 2014-10-27 | 2017-08-31 | The Governing Council Of The University Of Toronto | Microfluidic device for cell-based assays |
CN106811413A (zh) * | 2015-11-30 | 2017-06-09 | 中国科学院大连化学物理研究所 | 基于微流控技术的多器官芯片及其制备方法 |
WO2017175236A1 (en) * | 2016-04-06 | 2017-10-12 | Dandekar Jain Prajakta | Microfluidic platform for developing in-vitro co-cultures of mammalian tissues. |
CN107502547A (zh) * | 2017-09-25 | 2017-12-22 | 中科芯瑞(苏州)生物科技有限公司 | 一种实现多种细胞共培养的微流控芯片及其应用 |
CN108641931A (zh) * | 2018-04-04 | 2018-10-12 | 浙江大学 | 一种数字化微阵列器官芯片及其应用 |
Non-Patent Citations (1)
Title |
---|
TINGTING TAO等: "Engineering human islet organoids from iPSCs using an organ-on-chip platform", 《LAB ON A CHIP》, 23 January 2019 (2019-01-23), pages 1 - 16 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112553076A (zh) * | 2020-12-15 | 2021-03-26 | 东南大学 | 脑类器官体外培养芯片 |
TWI751902B (zh) * | 2021-01-29 | 2022-01-01 | 錢家琪 | 細胞培養平台及其製造方法 |
CN113667633A (zh) * | 2021-07-29 | 2021-11-19 | 四川大学华西医院 | 一种精神分裂症发育异常的皮层类器官模型的构建方法 |
CN114736798A (zh) * | 2022-04-13 | 2022-07-12 | 哈尔滨工业大学 | 一种基于软性材料的胰岛β细胞微型提取模具和提取方法 |
CN114849801A (zh) * | 2022-04-26 | 2022-08-05 | 复旦大学 | 通量化体外细胞、组织、器官培养和分析的微流控装置 |
CN117363482A (zh) * | 2023-12-06 | 2024-01-09 | 中国医学科学院北京协和医院 | 一种用于不同种类的类器官联合培养的方法 |
CN117363482B (zh) * | 2023-12-06 | 2024-03-29 | 中国医学科学院北京协和医院 | 一种用于不同种类的类器官联合培养的方法 |
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