CN117004559A - 一种人多能干细胞来源的脑和心肌复合模型的构建方法 - Google Patents
一种人多能干细胞来源的脑和心肌复合模型的构建方法 Download PDFInfo
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Abstract
本发明涉及一种人多能干细胞来源的脑和心肌复合模型的构建方法。所述复合模型包括至少两个细胞培养室，所述细胞培养室的底部具有呈阵列排布的微坑结构或柱状结构以有利于细胞三维生长，所述细胞培养室通过多孔膜与上层培养基连通，实现间接连接；将多能干细胞诱导化的三维脑和心肌类器官接种到不同培养室中，能够实现两种类器官间代谢产物和培养基的连通和交换，从而促进两种组织间的共培养和互作，模拟体内脑和心脏的功能互联。本发明所述复合模型的结构在满足两种器官共培养的同时，能够有效降低因灌注培养产生流体剪切力对类器官发育和功能的影响。
Description
技术领域
本发明属于干细胞及组织工程领域，涉及一种多能干细胞来源的人脑和心肌共培养的复合模型，具体涉及一种基于器官芯片技术的多能干细胞来源的人脑和心肌类器官的共培养体系构建方法。
背景技术
脑心综合征是由脑出血、蛛网膜下腔出血、急性颅脑外伤等急性脑损伤诱发的心律失常、心肌损伤的统称。研究显示，急性脑损伤时机体处于应激状态，HPA轴活性增强，促肾上腺皮质激素刺激肾上腺皮质释放皮质醇，同时交感神经活动增强，儿茶酚胺及神经肽相关递质释放增加。血清中持续的高儿茶酚胺状态可引经引起心肌损伤，包括收缩带坏死、水肿、收缩功能减弱，并与QT间期延长、T波改变相关；同时作用于冠状动脉使其痉挛与收缩，造成心肌缺血。急性脑损伤后，如神经肽Y、血栓素A2、前列腺素2a、内源性阿片肽等，一方面通过强烈的收缩血管作用加重心脏负担，另一方面直接抑制乳酸转运和能量代谢，导致心肌损伤。总体而言，脑心综合征疾病机制并不清楚，脑-心肌间的互作关系在疾病发生中作用并不清楚。现阶段，仍缺少组织水平研究器官间互作的体外模型体系。
近年来，干细胞技术的飞速发展，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。目前，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。但基于此技术的临床治疗还寥寥无几，其主要因素为：干细胞来源的细胞分化水平较低，处于不成熟阶段，且分化纯度不高。因此，寻找一种方法可促进心肌细胞更成熟、纯度更高的方法是亟待解决的问题。
已有研究结果表明，单纯混合培养脑和心肌细胞对研究单纯心肌和脑功能存在巨大限制，而Transwell等传统二维共培养难以构建三维多组分的脑和心肌类器官。因此，需要研发一种通量高、成本低、操作简单的三维非接触式复合器官芯片共培养体系。
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、脑等微器官模型。如何构建多器官共培养的培养基及培养环境是目前传统干细胞来源类组织器官面临的重大挑战。
发明内容
本发明目的在于提供一种人多能干细胞来源的脑和心肌复合模型的构建方法。在本发明所述的人脑和心肌类器官的共培养体系中，心肌细胞和脑细胞通过所分泌细胞因子相互促进，实现了体外长期心肌和脑的共培养。同时，在共培养体系中更有利于心肌和脑细胞活性和分泌功能。
为实现上述目的，本发明采用的技术方案为：
一种复合模型，所述复合模型包括至少两个细胞培养室，所述细胞培养室的底部具有呈阵列排布的微坑结构或柱状结构以有利于细胞三维生长，所述细胞培养室通过多孔膜与上层培养基连通，实现间接连接；将多能干细胞诱导化的三维脑和心肌类器官接种到不同培养室中，能够实现两种类器官间代谢产物和培养基的连通和交换，从而促进两种组织间的共培养和互作，模拟体内脑和心脏的功能互联。
所述复合模型包括上层、中间层和下层，所述上层为带有微通道结构的PDMS，所述中间层为多孔膜，所述下层为带有微坑结构的PDMS。
所述上层包括培养基入口、培养基出口、细胞入口、细胞出口，所述下层包括细胞入口、细胞出口、呈阵列排布的微坑结构；所述培养基入口与培养基出口相连形成上层培养基灌注通道，所述上层细胞入口与下层细胞入口连通形成完整细胞入口，所述上层细胞入口与下层细胞入口连通形成完整细胞入口，所述上层细胞出口与下层细胞出口连通形成完整细胞出口；
所述完整细胞入口经由所述微坑结构与所述完整细胞出口连通形成独立组织细胞培养室，所述完整细胞入口经由所述微坑结构与所述完整细胞出口连通形成独立组织细胞培养室。
所述微坑结构的深度为0.4-1.5mm，半径为0.2-0.4mm；
所述柱状结构的高度为0.4-1.5mm，半径为0.2-0.4mm。
所述培养基入口和培养基出口与灌流管连接。
所述培养基入口与培养基注入管连接，所述培养基出口与培养基输出管连接，所述培养基注入管与培养基输出管在同一培养基储存瓶内，形成闭合灌流体系；
所述培养基的注入流速为10-20微升每分钟。
所述多孔膜的孔径为1-50微米。
所述多孔膜为PDMS膜、聚碳酸酯膜或硝酸纤维素膜中的任一种。
一种基于所述复合模型的多能干细胞来源的人脑和心肌共培养的方法，包括以下步骤：
(1)脑和心肌细胞的接种
共培养基由所述复合模型上层的培养基入口进入，经由上层通道由培养基出口流出；
共培养实验中，将人诱导多能干细胞生成的脑和心肌前体细胞分散消化成单细胞，之后接种于细胞培养室；
(2)脑类器官的诱导分化
脑类器官单细胞接种后，先进行神经诱导分化，再向大脑各个皮层诱导分化及神经细胞分化；
(3)心肌类器官的诱导分化
心肌类器官单细胞接种后，先形成拟胚体，再进行诱导分化；
对诱导分化后的类器官进行功能鉴定，确定经基因、蛋白和分泌水平鉴定具有脑和心肌的部分生理功能。
用于诱导脑和心肌的多能干细胞包括胚胎干细胞和诱导多能干细胞。
所述三维组织水平的人源类脑和类心肌的大小为0.2-0.5mm。
本发明具有如下优点：
(1)本发明所述复合模型，通过在上层和下层之间设置多孔膜作为中间层，能够实现上层培养基通道的液体与下层培养室中液体的交换，提供培养室中类器官的营养物质和氧气。同时，该设计能够使两种类器官间接连通，从而实现功能互联。进一步的，本发明所述复合模型的结构在满足两种器官共培养的同时能够有效降低因灌注培养产生流体剪切力对类器官发育和功能的影响。
(2)本发明所述的复合模型，为一种多器官三维共培养体系，适用于其他三维类器官共培养，应用前景广泛，操作简单。
(3)本发明所述的复合模型，能够良好的模拟体内生理环境，有利于细胞体外功能的维持，脑和心肌类器官共培养，能够使脑(心肌)类器官产生的细胞因子等生物信号分子作用到连通的心肌(脑)类器官上，促进两种类器官的功能和活性，从而实现功能互联。
(4)高通量的三维共培养体系应用前景广泛。能满足针对心肌和脑互联的疾病，病理感染和环境暴露研究。结合高通量，低成本和自动化应用需求，将该芯片发展研制成为一种高通量、自动化、可集成多种生物传感器的装置，为推动相关药物研究的实际应用，提供更加快捷、精准、集约的测试平台，这将大大减少动物实验成本，助力新药研发领域。
(5)本发明所述的人脑和心肌共培养的方法，通过将类器官与器官芯片前沿技术相结合，从而特色性构建了一种由人多能干细胞衍生的脑-心肌类器官互作体系，能够再现人体脑-心肌互作介导生理和病理的响应，突破了现有传统研究手段的局限，为相关病研究和相关药物开发等提供了全新的策略、技术和思路。
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，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，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，还可以根据这些附图获得其他的附图。
图1A是本发明所述复合模型的整体结构示意图；
图1B是本发明所述复合模型的拆解结构示意图；
图中，1-带有微通道结构的PDMS，2-多孔膜，3-带有微坑结构的PDMS，4-培养基入口，5-培养基出口，6-细胞入口，7-细胞入口，8-细胞入口，9-细胞入口，10-细胞出口，11-细胞出口，12-细胞出口，13-细胞出口，14-呈阵列排布的微坑结构，15-上层培养基灌注通道，16-完整细胞入口，17-完整细胞出口，18-完整细胞入口，19-完整细胞出口，20-独立组织细胞培养室，21-独立组织细胞培养室；
图2A是本发明所述复合模型上诱导分化和产生的高通量类脑组织；
图2B是类脑组织的形态学表征；
图2C是免疫荧光鉴定Nestin、SOX2、PAX2、PAX6显示脑组织不同细胞分布；
图3A是经过诱导分化后，利用免疫荧光标记神经元特异标志蛋白TUJ1，表征神经突起相互连接形成的神经网络；
图3B是经过诱导分化后免疫荧光鉴定神经元特异标志蛋白TUJ1，在神经元胞体的表达；
图4A是芯片上高通量类心肌组织的诱导分化和产生；
图4B是类心肌组织切片染色标志物c-TNT鉴定；
图5是通过调节干细胞密度形成不同形状的类心肌微组织。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。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的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
实施例1
本实施例提供一种诱导多能干细胞来源的人脑和心肌类器官共培养的复合模型，结构如图1A和图1B所示，包括两个细胞培养室，所述细胞培养室的底部具有呈阵列排布的微坑结构以有利于细胞三维生长，所述细胞培养室之间由多条微通道串联连接。所示复合模型包括上层、中间层和下层，上层为带有微通道结构的PDMS 1，中间层为多孔膜2，下层为带有微坑结构的PDMS 3。
作为优选的实施方式，本实施例中，所述上层带有微通道结构的PDMS结构的长为25mm，宽为5mm，高为0.5mm；所述微坑结构的深度为0.8mm，半径为0.2mm，微坑结构的个数为60个。
所述上层结构设有培养基入口4、培养基出口、细胞入口6和8、细胞出口10和12，所述下层结构设有细胞入口7和9、细胞出口11和13、呈阵列排布的微坑结构14；所述培养基入口4和培养基出口5相连形成上层培养基灌注通道15，所述上层细胞入口6与下层细胞入口7连通形成完整细胞入口16，所述上层细胞入口8与下层细胞入口9连通形成完整细胞入口18，所述上层细胞出口10与下层细胞出口11连通形成完整细胞出口19。
所述完整细胞入口16经由所述微坑结构14与所述完整细胞出口17连通形成独立组织细胞培养室20，所述完整细胞入口18经由所述微坑结构14与所述完整细胞出口19连通形成独立组织细胞培养室21。
进一步，本实施例提供一种基于所述复合模型的诱导多能干细胞来源的人脑和心肌共培养的方法，用来共培养的脑和心肌是利用多能干细胞诱导化的三维组织水平的人源类脑和类心肌，经基因、蛋白和分泌水平鉴定具有脑和心肌的部分生理功能。
所述人脑和心肌共培养的方法具体包括以下步骤：
(1)脑和心肌细胞的接种
共培养基由所述复合模型上层的培养基入口4进入，经由上层通道15由培养基出口5流出；共培养基成分为DMEM(Invitrogen)添加0.5M N-乙酰半胱氨酸(R&D)、1％B27(Invitrogen)、1％N2(Invitrogen)、1％GlutaMAX(Invitrogen)、1％非必需氨基酸(NEAA，Invitrogen)和1％青霉素-链霉素(Invitrogen)；
共培养实验中，将6孔板中的人诱导性多潜能干细胞(hiPSCs)生成的心肌和脑前体细胞用Dispase分散酶(1ml/孔)消化成单细胞，以5*103每毫升的密度接种于细胞培养室20、21；
心肌细胞由完整细胞入口16接种，脑细胞由细胞入口18接种；
(2)脑类器官的诱导分化：
脑类器官诱导分化分为两个阶段，第一阶段为神经诱导分化阶段：去除mTeSR1，加入2ml第一阶段诱导培养基，静止培养5-6天；每2天换一次液；
所述第一阶段诱导培养基的基础成分为DMEM/F12，加入占总体积0.8-1％的N2，占总体积0.8-1％的GlutaMAX，占总体积0.8-1％的MEM-NEAA，终浓度为1-2μg/ml的heparin；占总体积1％penicillin-streptomycin(100×)，终浓度10μg/ml抗支原体药物，终浓度10μg/ml Gentamicin，终浓度0.25μg/ml Amphotericin B。
第二阶段主要向大脑各个皮层诱导分化以及神经细胞分化，第二阶段的培养基基础成分为DMEM/F12，另外需添加占总体积50％的Neurobasal medium，加入占总体积0.5-1％N2，占总体积0.5-1％B27，占总体积0.8-1％GlutaMAX，占总体积0.8-1％MEM-NEAA，占总体积0.3-0.4‰β-mercaptoethanol；占总体积1％penicillin-streptomycin(100×)，终浓度10μg/ml抗支原体药物，终浓度10μg/ml Gentamicin，终浓度0.25μg/ml AmphotericinB。
(3)心肌类器官的诱导分化
步骤(1)处理后的心肌类器官接种于培养室中，待细胞球自然沉降之后，培养24小时，形成拟胚体，再加入2ml诱导分化培养基1640/B27-insulin，添加终浓度在6-12μM的CHIR99021作用24-30小时；以加入CHIR99021作为诱导分化第1天，第2、3天分别加入2ml诱导培养基1640/B27-insulin，第4、5天分别加入2ml诱导分化培养基1640/B27-insulin，添加终浓度6-12μM的IWP2；第7天以后加入2ml心肌细胞培养基1640/B27，每隔1天更换新鲜心肌细胞培养基。以10微升每分钟的灌注速度进行培养。在培养期间，每隔5天进行细胞活性和功能鉴定。
将已经诱导的类器官进行功能鉴定，图2A和图2B分别显示高通量的类脑组织的形成，所形成的类脑组织大小为0.2-0.5mm。图2C为免疫荧光鉴定Nestin、SOX2、PAX2、PAX6显示脑组织不同细胞分布。图3A是经过诱导分化后神经突起相互连接形成的神经网络，图3B是免疫荧光鉴定神经元特异标志蛋白TUJ1。
图4A显示芯片上高通量的类心肌微组织，所形成的类心肌组织大小为0.2-0.5mm。利用心肌细胞特定标志蛋白cTnT免疫荧光染色，如图4B所示，阳性染色组织为心肌组织，结果显示90％以上的拟胚体cTnT表达阳性，白色箭头为cTnT阳性表达的心肌组织。
对复合芯片心肌类器官进行功能鉴定。通过调节干细胞密度，可以形成多种形状的类心肌微组织，如5中所示。
实施例2
本实施例与实施例1的区别仅在于，所述细胞培养室的底部具有呈阵列排布的微柱结构以有利于细胞三维生长。所述柱状结构的高度为0.4-1.5mm，半径为0.2-0.4mm。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.一种复合模型，其特征在于，所述复合模型包括上层、中间层和下层，所述上层为带有微通道结构的PDMS(1)，所述中间层为多孔膜(2)，所述下层为带有呈阵列排布的微坑结构或柱状结构的PDMS(3)；
所述复合模型包括至少两个细胞培养室，所述细胞培养室通过多孔膜与位于上层的培养基连通；将多能干细胞诱导化的三维脑和心肌类器官接种到不同细胞培养室中，能够实现两种类器官间代谢产物和培养基的连通和交换。
2.根据权利要求1所述的复合模型，其特征在于，所述上层包括培养基入口(4)、培养基出口(5)、细胞入口(6)(8)、细胞出口(10)(12)，所述下层包括细胞入口(7)(9)、细胞出口(11)(13)、呈阵列排布的微坑结构(14)；所述培养基入口(4)与培养基出口(5)相连形成上层培养基灌注通道(15)，所述上层细胞入口(6)与下层细胞入口(7)连通形成完整细胞入口(16)，所述上层细胞入口(8)与下层细胞入口(9)连通形成完整细胞入口(18)，所述上层细胞出口(10)与下层细胞出口(11)连通形成完整细胞出口(19)；
所述完整细胞入口(16)经由所述微坑结构(14)与所述完整细胞出口(17)连通形成独立组织细胞培养室(20)，所述完整细胞入口(18)经由所述微坑结构(14)与所述完整细胞出口(19)连通形成独立组织细胞培养室(21)；
所述复合模型的培养室内能够接种类脑、类心肌中的任何一种。
3.根据权利要求1所述的复合模型，其特征在于，所述微坑结构的深度为0.4-1.5mm，半径为0.2-0.4mm；
所述柱状结构的高度为0.4-1.5mm，半径为0.2-0.4mm。
4.根据权利要求1所述的复合模型，其特征在于，所述培养基入口(4)和培养基出口(5)与灌流管连接。
5.根据权利要求1所述的复合模型，其特征在于，所述培养基入口(4)与培养基注入管连接，所述培养基出口(5)与培养基输出管连接，所述培养基注入管与培养基输出管在同一培养基储存瓶内，形成闭合灌流体系；
所述培养基的注入流速为10-20微升每分钟。
6.根据权利要求1所述的复合模型，其特征在于，所述多孔膜的孔径为1-50微米。
7.根据权利要求1所述的复合模型，其特征在于，所述多孔膜为PDMS膜、聚碳酸酯膜或硝酸纤维素膜中的任一种。
8.一种基于权利要求1-7任一所述复合模型的多能干细胞来源的人脑和心肌共培养的方法，其特征在于，包括以下步骤：
(1)脑和心肌细胞的接种
共培养基由所述复合模型上层的培养基入口(4)进入，经由上层通道(15)由培养基出口(5)流出；
共培养实验中，将人诱导多能干细胞生成的脑和心肌前体细胞分散消化成单细胞，之后接种于细胞培养室；
(2)脑类器官的诱导分化
脑类器官单细胞接种后，先进行神经诱导分化，再向大脑各个皮层诱导分化及神经细胞分化；
(3)心肌类器官的诱导分化
心肌类器官单细胞接种后，先形成拟胚体，再进行诱导分化；
对诱导分化后的类器官进行功能鉴定，确定经基因、蛋白和分泌水平鉴定具有脑和心肌的部分生理功能。
9.根据权利要求8所述的多能干细胞来源的人脑和心肌共培养的方法，其特征在于，用于诱导脑和心肌的多能干细胞包括胚胎干细胞和诱导多能干细胞。
10.根据权利要求8所述的多能干细胞来源的人脑和心肌共培养的方法，其特征在于，所述三维组织水平的人源类脑和类心肌的大小为0.2-0.5mm。
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| CN117384759A (zh) * | 2023-12-05 | 2024-01-12 | 中国医学科学院北京协和医院 | 一种基于微针阵列的类器官培养方法 |
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| CN117384759B (zh) * | 2023-12-05 | 2024-03-29 | 中国医学科学院北京协和医院 | 一种基于微针阵列的类器官培养方法 |
| 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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