CN113717925B - 一种人工肝脏类器官及其制备方法和应用 - Google Patents
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Abstract
本发明涉及生物材料和生物医学工程领域,尤其涉及一种人工肝脏类器官及其制备方法和应用。所述人工肝脏类器官包括:内皮细胞层、肝星状细胞层、肝实质细胞层;不同细胞层之间呈仿生紧密排布,相邻的不同种类细胞之间的间距小于30μm;所述人工肝脏类器官的力学性能为500Pa‑1MPa;所述人工肝脏类器官的特征长度为50‑1000μm。本发明构建的肝脏类器官包括肝细胞、内皮细胞和肝星状细胞,能够模拟肝脏Disse间隙的结构和生理功能,满足体外肝脏生理/病理学研究、药物测试与药物开发、组织工程和再生医学的要求。本发明提供的人工肝脏类器官同时可以诱导成为多种肝脏疾病模型,可根据需求构建对应的病理模型。
Description
技术领域
本发明涉及生物材料和生物医学工程领域,尤其涉及一种人工肝脏类器官及其制备方法和应用。
背景技术
肝脏是人体最大的内脏器官,承担毒性物质代谢、脂类和糖类代谢与合成等生理功能。肝脏Disse间隙是肝脏的生理结构,由肝实质细胞、肝窦内皮细胞、肝星状细胞和细胞外基质成分组成。肝实质细胞承担生物合成、脂类代谢和解毒等多种功能。肝窦内皮细胞形成的微血管结构是肝细胞和血液进行物质交换的场所,微血管中血流刺激在肝脏结构形成和功能成熟过程中发挥着重要的作用。肝星状细胞位于Disse间隙,紧贴着肝实质细胞和肝窦内皮细胞,生理状态下呈静息状态,受到刺激后转变成激活态。静息态的肝星状细胞能储存维生素A且能分泌肝脏生长因子促进肝细胞的成熟。当肝脏受到损伤,肝星状细胞由静息态转变成激活态,维生素A储存水平下降,分泌大量的胶原蛋白到细胞外基质中,从而改变细胞外基质的组成,甚至出现病理特征。
类器官指由多种干细胞通过细胞-细胞相互作用组装形成,或者由一种干细胞分化出的多种细胞在分化过程中自组装形成结构、功能与目标器官类似的细胞集合。目前基于肝脏干细胞、间充质干细胞、内皮细胞、肝细胞细胞系、肝星状细胞系、胚胎干细胞和诱导多能干细胞等构建肝脏类器官的研究多有报道,但肝脏Disse间隙类器官仍然稀缺。此外,目前类器官构建多采用大块水凝胶直接包埋的方法,此方法影响养分和氧气供给,造成类器官生产效率低下、均一性差等问题。
发明内容
为了解决现有技术本存在的问题,本发明提供一种人工肝脏类器官及其制备方法和应用,本发明构建包含肝实质细胞、肝星状细胞、肝窦内皮细胞且细胞比例可控的类器官模拟Disse间隙,以满足药物临床前检测、精准医疗、环境监测、毒理检测、组织工程、再生医学、新药研发、肝组织发育、肝脏病理学、研究疾病发生和发展等诸多领域的要求。
第一方面,本发明提供一种人工肝脏类器官,所述人工肝脏类器官包括:
内皮细胞层、肝星状细胞层、肝实质细胞层;不同细胞层之间呈仿生紧密排布,相邻的不同种类细胞之间的间距小于30μm;
所述人工肝脏类器官的力学性能为500Pa-1MPa;
所述人工肝脏类器官的特征长度为50-1000μm。
进一步地,所述人工肝脏类器官的特征长度为50-500μm。
进一步地,还包括水凝胶层;所述水凝胶层位于所述肝实质细胞层外侧。
目前学术界尚未有体外构建出disse间隙的报道。这主要是由于目前学术界对于类器官的制备关注点均在于细胞因子方面,希望通过对于细胞因子的系统研究和精确添加,达到体外制备disse间隙类器官的目的。申请人发现这类研究很多,但是都没有达到细胞排布出现仿生结构的程度,而且目前普遍使用的matrigel(基质胶)材料存在成分不明确以及批次差异大等问题。这就使得制备得到类器官材料力学性能较差、难以调控,并且物质在其中的扩散和梯度分布较差。本申请通过控制细胞种类和排布方式,力学性能和特征长度得到的人工肝脏类器官中,多种细胞可以顺利在体外环境中同步发育、成熟、组装,进而形成能够精准仿生disse间隙结构和细胞排布的人工器官。
所述内皮细胞层中的内皮细胞、所述肝星状细胞层中的肝星状细胞和所述肝实质细胞层中的肝实质细胞的数量比为10:(1~100):(1~100);和/或,
所述人工肝脏类器官的细胞密度为106~109个/mL。
所述水凝胶层由天然水凝胶材料和/或人工合成水凝胶材料组成;
所述天然水凝胶材料包括明胶或其衍生物、藻酸盐或其衍生物、纤维素或其衍生物、琼脂、基质胶、胶原或其衍生物、氨基酸或其衍生物、糖蛋白及其衍生物、透明质酸或其衍生物、壳聚糖或其衍生物、层连接蛋白、纤连接蛋白、纤维蛋白或其衍生物、丝素蛋白或其衍生物、玻连蛋白、骨桥蛋白、肽段水凝胶、DNA水凝胶中的一种或多种,优选为海藻酸钠、明胶、基质胶或胶原;
所述人工合成水凝胶材料包括聚丙烯、聚苯乙烯、聚丙烯酰胺、聚丙交酯、聚乙交酯、聚乳酸、聚乳酸-羟基乙酸共聚物、聚羟基酸、聚乳酸醇酸共聚物、聚二甲基硅氧烷、聚酸酐、聚酸酯、聚酰胺、聚氨基酸、聚缩醛、聚氰基丙烯酸酯、聚氨基甲酸酯、聚吡咯、聚酯、聚甲基丙烯酸酯、聚乙烯、聚碳酸酯或聚氧化乙烯等中的一种或多种,优选为聚乳酸或乳酸-羟基乙酸共聚物。
进一步地,所述肝实质细胞层中的肝实质细胞的来源包括如下任意一种或多种:
i)胚胎干细胞、诱导多能干细胞、肝脏干细胞、肝脏祖细胞、内胚层细胞、肝脏内胚层细胞、肝母细胞、间充质干细胞、成体干细胞、肝细胞或肝样细胞;
ii)由i)经过基因编辑、病毒包装、建系或改造获得的细胞;
和/或,
所述内皮细胞层中的内皮细胞的来源包括如下任意一种或多种:
iii)胚胎干细胞、诱导多能干细胞、内皮祖细胞、内皮干细胞、中胚层细胞、间充质干细胞、成体干细胞、内皮细胞或内皮样细胞;
iv)由iii)经过基因编辑、病毒包装、建系或改造获得的细胞;
所述肝星状细胞层中的肝星状细胞的来源包括如下任意一种或多种:
v)胚胎干细胞、诱导多能干细胞、中胚层细胞、间质细胞、间皮细胞、间充质干细胞、成体干细胞、肝星状细胞或肝星状样细胞;
vi)由v)经过基因编辑、病毒包装、建系或改造获得的细胞。
本发明中使用的肝实质细胞、内皮细胞和肝星状细胞可以来源于多种具有分化为相应细胞潜力的细胞,例如上述的胚胎干细胞、诱导多能干细胞等。
进一步地,所述肝实质细胞层中的肝实质细胞来源于诱导多能干细胞或肝脏干细胞分化得到的肝细胞;
所述内皮细胞层中的内皮细胞来源于诱导多能干细胞或内皮组细胞分化得到的内皮细胞;
所述肝星状细胞层中的肝星状细胞来源于诱导多能干细胞或胚胎干细胞分化得到的肝星状细胞。
进一步地,在诱导多能干细胞分化为肝细胞,使用的诱导培养基中添加B27和/或血清替代物,此外添加细胞因子,包括30-300ng/mL Activin A、10-500ng/mL Wnt3A、10-500ng/mL BMP4、5-500ng/mL FGF2、10-500ng/mL HGF、10-500ng/mL OSM、1-50μg/ml胰岛素和1-50μg/ml转运蛋白中的一种或多种,诱导时间为15~35天,可以获得肝细胞。
进一步地,在诱导间充质干细胞分化内皮细胞时,使用的培养基中添加B27和/或血清替代物,同时添加细胞因子,包括10-500ng/mL Activin A、10-500ng/mL Wnt3A、10-500ng/mL BMP4,5-500ng/mL FGF2,100-1000ng/mL VEGF、1-50μg/ml胰岛素、1-50μg/ml转运蛋白中的一种或多种,诱导时间为10~30天,可以获得内皮细胞。
进一步地,在诱导胚胎干细胞分化为肝星状细胞,使用的培养基中添加细胞因子,包括5-400ng/mL BMP4,5-500ng/mL FGF2,5-500ng/mL FGF3,5-50ng/mL IGF-1中的一种或多种,诱导时间为1~30天,可以获得肝星状细胞。
进一步地,所述人工肝脏类器官还包括胆管上皮细胞、成纤维细胞或枯否细胞。
进一步地,所述人工肝脏类器官的形状为类球状体、类柱状体或类多边棱柱体中的一种或多种。
进一步地,所述人工肝脏类器官的杨氏模量为0.1-200kPa。
第二方面,本发明提供所述人工肝脏类器官的制备方法,包括:
(1)制备肝实质细胞、内皮细胞和肝星状细胞;
(2)将所述肝实质细胞、内皮细胞和肝星状细胞与水凝胶材料混合;
(3)通过工程化方法使步骤(2)得到的混合物三维成型,得到水凝胶3D结构体;
(4)将步骤(3)得到的水凝胶3D结构体进行培养,使其中的细胞自组装得到所述人工肝脏类器官。
所述水凝胶3D结构体优选为球状体、丝状体、棱柱体、柱状、块状、片状、囊状、管状、网络状、编织状中的任意一种或多种。
进一步地,还包括后处理流程;
所述后处理流程为稳定化处理和/或牺牲材料处理;
所述稳定化处理使用的交联试剂为二价阳离子、凝血酶、京尼平、戊二醛、已二酸二酰肼、环氧氯丙烷、碳化二亚胺或其衍生物中的一种或多种,优选为二价阳离子和/或凝血酶。
进一步地,所述交联试剂的浓度为0.1mM~10M,优选10mM~500mM。
所述牺牲材料处理包括去除多余材料,所述多余材料包括三维水凝胶结构体中的温敏材料(如明胶、胶原蛋白、N-异丙基丙烯酰胺和聚乙烯吡咯烷酮等)、交联试剂、光引发剂等。
进一步地,步骤(4)中的培养包括静态培养、动态培养或大规模细胞培养;
所述静态培养在培养皿、培养瓶或多孔板中进行;
所述动态培养在气升式生物反应器、鼓泡式生物反应器、中空纤维生物反应器、陶质矩形通道蜂窝状反应器、玻璃珠床反应器、流化床反应器、固化床反应器、脉动培养装置、微重力培养装置、搅拌培养装置、波浪式培养装置、芯片或灌注等培养系统中进行;
所述大规模细胞培养包括NUNC细胞工厂、良方人工智能辅助全自动扩增设备等各种形式的细胞大规模扩增平台和装置。
进一步地,步骤(4)中细胞培养基包括血清替代物以及细胞因子;所述细胞因子包括10-500ng/mL HGF,10-500ng/mL OSM,5-500ng/mL VEGFA,5-200ng/mL FGF2中任意一种或多种。
进一步地,步骤(4)中细胞培养时间为3~30天,优选为5~15天。
本发明进一步提供所述人工肝脏类器官作为肝脏类实验模型中的应用;药物临床前检测、面向肝脏疾病的精准医疗、环境/毒性物质/污染物监测、肝脏毒理检测、肝组织工程与再生医学、肝脏疾病相关新药研发、肝脏发育生理学、肝脏病理学或药物毒性和药效试验。
本发明具备如下有益效果:
1、本发明提供的水凝胶结构体能够模拟生理环境下细胞外基质的结构和功能,提供稳定且仿生的三维微环境。微结构体的合适尺寸与水凝胶材料的多孔性保证了细胞和培养环境的良好物质交换,利于细胞在微结构体内的长期稳定生存。生物材料为细胞提供了附着点,细胞可以在微结构内进行粘附、迁移,有利于细胞的空间排布及组装。
2、本发明构建的肝脏类器官能够一定程度上模拟肝脏生理结构和功能。本发明构建的肝脏类器官包括肝细胞、内皮细胞和肝星状细胞,能够模拟肝脏Disse间隙的结构和生理功能,满足体外肝脏生理/病理学研究、药物测试与药物开发、组织工程和再生医学的要求。
3、工程化方法制备微结构效率和可重复性高,满足毒理学研究高通量的要求,且能够在不同平台实现快速技术转化。本发明使用工程化方法,制造工艺可规范化,大幅度降低人为操作引起的差异,且生产效率高,能大规模获得水凝胶类器官。本发明构建的肝脏类器官细胞类型、比例可控,形成的类器官在批次间和批次内中的差异较小,可以满足药物毒理学和环境毒理学研究对实验模型仿可重复性高、高通量、背景噪音低的要求。
4、本发明提供的人工肝脏类器官的制备方法可以定制结构高保真的水凝胶肝脏类器官模型,可以使用病人来源的干细胞构建肝脏类器官,用于精准医疗和药物研发。本发明构建的肝脏类器官生理模型可以诱导成为多种肝脏疾病模型,可根据需求构建对应的病理模型。
附图说明
图1为本发明实施例1提供的人工肝脏类器官示意图;其中1为肝实质细胞,2为水凝胶材料,3为内皮细胞,4为肝星状细胞。
图2为本发明实施例1提供的iPSC分化的限制性内胚层细胞、肝脏内胚层细胞、肝祖细胞、肝实质细胞的免疫荧光染色图;图片在10倍镜下拍摄。
图3为本发明实施例1提供的iPSC分化的内皮祖细胞和内皮细胞的免疫荧光染色图;图片在10倍镜下拍摄。
图4为本发明实施例1提供的iPSC分化的中胚层细胞、肝星状祖细胞和肝星状细胞的免疫荧光染色图;图片在10倍镜下拍摄。
图5为本发明实施例1提供的工程化方法三维成形后,水凝胶微结构体中细胞存活染色图;图片为在10倍镜下拍摄的三维最大投影图。
图6为本发明实施例1提供的工程化方法三维成形后,不同天数的肝脏类器官的形貌;其中A是成形后第3天;B是成形后第5天;C是成形后第10天。
图7为本发明实施例1提供的工程化方法三维成形后第15天,水凝胶内肝脏类器官标志基因表达情况,对照组为同样的种子细胞在同样培养液条件下2D平面培养情况;肝脏类器官组基因相对表达值是以对照组进行均一化计算。
图8为本发明实施例1提供的工程化方法三维成形后第15天,肝脏类器官的关键标志物染色;A是内皮标志蛋白CD31和肝实质细胞标志蛋白ALB免疫荧光染色图;B是肝实质细胞标志蛋白ALB和肝星状标志蛋白PDGFRB免疫荧光染色图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1采用静电生物打印技术工程化制备含有多种细胞的水凝胶3D结构体
本实施例提供一种制备人工肝脏类器官(如图1所示,其中1为肝实质细胞,2为水凝胶材料,3为内皮细胞,4为肝星状细胞)的流程,具体如下:
1、诱导性多能干细胞(iPSC)定向分化为肝实质细胞
(1)细胞分化:
将iPSC(ATCC)培养至细胞汇合80%时开始诱导分化;
先采用诱导培养基1培养3-5天,成分包括DMEM-F12(Thermo Fisher),1×B27(Gibco),50ng/mL Activin A(R&D,338-AC),此阶段可获得限制性内胚层细胞。
接下来采用诱导培养基2培养4-8天,成分包括DMEM-F12(Thermo Fisher),B27(Gibco),200ng/mL BMP4(PeproTech),50ng/mL FGF2(R&D),此阶段可获得肝脏内胚层细胞。
接下来采用诱导培养基3培养4-12天,成分包括DMEM-F12(Thermo Fisher),50ng/mL HGF(R&D systems)。此阶段可获得肝祖细胞。
接下来采用诱导培养基4培养4-10天,成分包括DMEM-F12(Thermo Fisher),2%血清替代物(BI),100μM Hydrocortisone(Sigma),30μg/ml胰岛素(Sigma),30μg/ml转运蛋白(Sigma),0.1%BSA(Sigma,9048-46-8),10ng/mL HGF(R&D systems)和20ng/mL OSM(R&Dsystems)。此阶段获得肝实质细胞。
(2)细胞形态观察与生物学检测:
使用细胞免疫荧光染色鉴定不同分化阶段细胞的标志蛋白的表达,使用激光扫描共聚焦显微镜观察记录,结果如图2所示(SOX17和FOXA2是限制性内胚层标志蛋白;HNF4A和FOXA2是肝脏内胚层标志蛋白;HNF4A和AFP是肝祖细胞标志蛋白;ALB和CYP3A4是肝实质细胞标志蛋白)。iPSC向肝实质细胞分化过程中,限制性内胚层标志蛋白SOX17和FOXA2均高表达,肝脏内胚层标志蛋白HNF4A和FOXA2高表达,且分化效率高达95%以上,这表明了本发明可以获得HNF4A和AFP阳性的肝脏祖细胞和ALB和CYP3A4阳性的肝实质细胞。
2、间充质干细胞定向分化为内皮细胞
(1)细胞分化:
将间充质干细胞(ATCC)培养至细胞汇合90%时开始诱导分化;
先采用诱导培养基1培养2-4天,成分包括RPMI1640(life),B27(Gibco),50ng/mLActivin A(R&D),400ng/mL BMP4(PeproTech),促进间充质细胞向内皮族系的分化趋势;
接下来采用诱导培养基2培养4-12天,成分包括DMEM-F12(Thermo Fisher),1×GlutaMax(Thermo Fisher),0.5%BSA(Sigma),300ng/mL VEGFA(R&D),50ng/mL FGF2(PeproTech),10ng/mL BMP4(PeproTech),25μg/ml胰岛素(Sigma)和50μg/ml转运蛋白(Sigma)。此阶段可获得内皮祖细胞;
接下来采用诱导培养基3培养4-14天,成分包括DMEM-F12/M199(Thermo Fisher),20μg/ml胰岛素(Sigma),5μg/ml转运蛋白(Sigma),1×GlutaMax(Sigma),2%FBS(Bioind),20ng/mL FGF2(PeproTech),300ng/mL Wnt3a(R&D)。此阶段获得内皮细胞。
(2)细胞形态观察与生物学检测:
使用细胞免疫荧光染色鉴定不同分化阶段细胞的标志蛋白的表达,使用激光扫描共聚焦显微镜观察记录,结果如图3所示(CD34是内皮祖细胞标志蛋白;CD31和CD32B是内皮细胞标志蛋白)。内皮祖细胞高表达标志蛋白CD34,内皮细胞高表达标志蛋白CD31和CD32B。
3、胚胎干细胞(ESCs)定向分化为肝星状细胞
(1)细胞分化:
将ESCs培养至细胞汇合50%时开始诱导分化。
先采用诱导培养基1培养3-7天,成分包括RPMI1640(life),250mg/L BSA(Sigma),50μg/mL Vitamin C(Sigma),25μM地塞米松(Sigma),20ng/mL BMP4(PeproTech)。
接下来采用诱导培养基2-8天,成分包括DMEM-F12(Thermo Fisher),1×GlutaMax(Thermo Fisher),50mg/L BSA(Sigma),50μM地塞米松(Sigma),200ng/mL FGF3(biolegend),200ng/mL BMP4(PeproTech)。此阶段获得中胚层细胞。
接下来采用诱导培养基3培养2-8天,成分包括DMEM-F12/M199(Thermo Fisher),200μg/ml胰岛素(Sigma),50μg/ml转运蛋白(Sigma),25μM地塞米松(Sigma),200ng/mLFGF3(Sigma),50μM retinol(Sigma),100pM Palmitic Acid(Sigma。此阶段获得肝星状祖细胞。
接下来采用诱导培养基4培养3-7天,成分包括DMEM-F12/M199(Thermo Fisher),250mg/L BSA,500μg/mL Vitamin C,250μM地塞米松,50μM retinol。此阶段获得肝星状细胞。
(2)细胞形态观察与生物学检测:
使用细胞免疫荧光染色鉴定不同分化阶段细胞的标志蛋白的表达,使用激光扫描共聚焦显微镜观察记录,结果如图4所示(KDR是中胚层标志蛋白;NCAM和ALCAM是肝星状祖细胞标志蛋白;PDGFRB是肝星状细胞标志蛋白),本实施例获得KDR阳性表达的中胚层细胞,NCAM和ALCAM阳性表达的肝星状祖细胞和PDGFRB阳性表达的肝星状细胞。
4、细胞-水凝胶悬液的制备
将步骤1-3获得的3种细胞,用胰蛋白酶(Sigma)消化3分钟,使其消化成单细胞后收集细胞沉淀。将海藻酸钠溶液、胶原溶液和细胞悬液均匀混合,获得细胞-水凝胶悬液,其中海藻酸钠浓度为1.5%,胶原浓度为100μg/ml,细胞密度为3×105cells/ml,肝细胞、内皮细胞和肝星状细胞的数量比例是1:1:1。
5、水凝胶3D结构体的制备
(1)三维成形
将步骤4获得的细胞-水凝胶悬液使用高压静电法打印细胞微结构(具体参见YaoR,Zhang R,Luan J,et al.Alginate and alginate/gelatin microspheres for humanadipose-derived stem cell encapsulation and differentiation.Biofabrication,2012,4(2):025007),直流高压电源电压设置为10KV,注射泵推进速度30ml/h,电极间距离为3cm,接收皿中加入400mM CaCl2溶液。细胞-水凝胶微滴落入接收皿并交联形成三维微结构体。
(2)细胞活死染色检测:
使用2μM Calcein-AM(Dojindo,C326)和4.5μM PI(Dojindo,P346)的混合溶液分别对活(绿色)/死(红色)细胞进行染色,染色避光进行,持续15分钟。使用激光扫描共聚焦显微镜(LSCM,Nikon,Z2)观察记录。对活死染色的照片进行数据统计,如图5所示,工程化方法三维成形后,微结构体内细胞存活率约95%左右。
6、细胞共培养形成类器官及其检测
(1)将步骤5获得的微结构体培养在多孔板中,使多种细胞在微结构体提供的三维环境中自发组装,形成仿生肝脏类器官。细胞共培养基成分包括,DMEM-F12(ThermoFisher),5μM monothioglycerol(Sigma-Aldrich),1×B27(Gibco),500μg/mL Vitamin C(Sigma-Aldrich),0.1%BSA(Sigma),1%sodium pyruvate(Sigma),1%NEAA(Sigma),200ng/mL VEGFA(R&D),200ng/mL HGF(R&D),50μg/mL FGF2(R&D)。
(2)形态观察:用光学显微镜(Olympus,CX40)每天观察细胞的状态。如图6所示(多种细胞包裹在水凝胶中三维培养,形成类器官),可以观察到第3天,水凝胶微结构中出现直径20-30μm细胞团(图6中的A),第5天细胞团逐渐变大(图6中的B),第10天细胞团的直径80-160μm(图6中的C)。
(3)生物学检测:
为了检测类器官中微血管的形成,肝实质细胞和肝星状细胞的功能成熟情况,采用qPCR技术检测肝实质细胞、肝星状细胞和内皮细胞相关基因(如CD31、ALB、CYP3A4、PDGFRβ、AFP、VIM)的转录水平。结果如图7所示,和2D分化相比,在3D条件下共培养的类器官,ALB、CYP3A4、CD31、PDGFRB、AFP和VIM基因表达水平显著上升,说明肝实质细胞、内皮细胞和肝星状细胞在3D培养条件下更为成熟。具体方法如下:
收集第15天的微结构体,溶于200mM柠檬酸钠和150mM氯化钠溶液的混合溶剂中,去除水凝胶结构并获得类器官细胞团。提取细胞RNA操作步骤:用PBS洗涤类器官1次,每个结构加入1ml Trizol(Gibco,15596026),反复吹打混匀,在室温静置10分钟,然后转移至1.5ml的EP管中,加入200μl氯仿,快速摇30秒,室温放置5分钟后,在4℃以12000g条件离心10分钟。去除上清液,加入等体积异丙醇,在4℃以12000g条件离心10分钟。弃去上清,用75%无水乙醇洗涤沉淀,风干后可获得RNA,使用DEPC水溶解。用spectrophotometer(Thermo Scientific)来检测RNA浓度及纯度。RNA反转录操作步骤:采用PrimeScriptTM II1st strand cDNA Synthesis Kit(TaKaRa,6210),完全按照试剂盒说明书来进行操作,RNA含量均调整为5ng,引物为Oligo dT Primer。使用PCR仪(ABI,SimpliAmpTM热循环仪)进行反转录,反应程序为:42℃50分钟,95℃5分钟,4℃保温。荧光定量PCR操作步骤:使用MaximaSYBR Green qPCR Master Mix(Thermo Scientific,K0251)试剂盒,完全按照试剂盒说明书进行操作。按要求加入反应液后,将反应板置于qPCR仪进行检测,反应程序为:95℃10分钟,95℃15s,60℃30s,40个循环;72℃30s,72℃10分钟。获得标志基因的表达情况。
qPCR所用引物序列如下(5′-3′):
ALB引物序列:
Forward:GCACAGAATCCTTGGTGAACAG,
Reverse:ATGGAAGGTGAATGTTTCAGCA;
CYP3A4引物序列:
Forward:TAACAGTCTTTCCATTCCTC,
Reverse:GGACTCAGTTTCTTTTGAAT;
CD31引物序列:
Forward:AAGTGGAGTCCAGCCGCATATC,
Reverse:ATGGAGCAGGACAGGTTCAGTC;
PDGFRβ引物序列:
Forward:TGCAGACATCGAGTCCTCCAAC,
Reverse:GCTTAGCACTGGAGACTCGTTG;
AFP引物序列:
Forward:GCAGAGGAGATGTGCTGGATTG,
Reverse:CGTGGTCAGTTTGCAGCATTCTG;
VIM引物序列:
Forward:AGGCAAAGCAGGAGTCCACTGA,
Reverse:ATCTGGCGTTCCAGGGACTCAT。
采用水凝胶细胞原位免疫荧光染色检测了内皮细胞、肝实质细胞和肝星状细胞特异性标记蛋白的表达(如CD31、ALB、PDGFRβ)。
免疫荧光染色方法如下:
收集第15天的微结构体,溶于200mM柠檬酸钠和150mM氯化钠溶液的混合溶剂中,去除水凝胶结构并获得类器官细胞团。4%多聚甲醛在室温下固定15分钟,用PBS洗涤3次,每次5分钟;
采用含0.3%Triton-X(Sigma,X100)和5%牛血清白蛋白(bovine serumalbumin,BSA)(Multicell,800-096-EG)的混合液封闭1小时;
吸出封闭缓冲液,加入稀释后的一抗(含0.1%Triton-X和1%BSA),4℃过夜孵育。用PBS洗涤3次,每次5分钟;
加入对应二抗(abcam,ab205718)和(abcam,ab205719),室温避光孵育2小时后,用PBS洗涤3次,每次5分钟;接着加入DAPI染细胞核,室温避光孵育5分钟。用激光共聚焦显微镜(LSCM,Nikon,Z2)观察记录。
结果如图8所示,内皮标志的蛋白CD31和肝实质细胞标志ALB共表达,ALB和PDGFRB共表达,说明多种细胞在水凝胶中三维培养成肝脏类器官。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 清华大学
<120> 一种人工肝脏类器官及其制备方法和应用
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Claims (4)
1.一种人工肝脏类器官的制备方法,其特征在于,包括:
(1)制备肝实质细胞、内皮细胞和肝星状细胞;
(2)将所述肝实质细胞、内皮细胞和肝星状细胞与水凝胶材料混合;
(3)通过工程化方法使步骤(2)得到的混合物三维成型,得到水凝胶3D结构体;所述水凝胶3D结构体为球状体、柱状、块状、片状、囊状、管状、网络状、编织状中的任意一种或多种;
(4)将步骤(3)得到的水凝胶3D结构体进行培养,使其中的细胞自组装得到所述人工肝脏类器官;
所述人工肝脏类器官由如下组成:内皮细胞层、肝星状细胞层、水凝胶层和肝实质细胞层;其中水凝胶层位于所述肝实质细胞层外侧;不同细胞层之间呈仿生紧密排布,相邻的不同种类细胞之间的间距小于30μm;所述人工肝脏类器官的力学性能为500Pa-1MPa;所述人工肝脏类器官的特征长度为50-1000μm;
所述内皮细胞层中的内皮细胞、所述肝星状细胞层中的肝星状细胞和所述肝实质细胞层中的肝实质细胞的数量比为10:(1~100):(1~100);所述人工肝脏类器官的细胞密度为106~109个/mL;
所述肝实质细胞层中的肝实质细胞来源于诱导性多能干细胞;
所述内皮细胞层中的内皮细胞来源于间充质干细胞;
所述肝星状细胞层中的肝星状细胞来源于胚胎干细胞。
2.根据权利要求1所述的制备方法,其特征在于,所述水凝胶层由天然水凝胶材料和/或人工合成水凝胶材料组成;所述天然水凝胶材料为海藻酸钠、明胶、基质胶或胶原;所述人工合成水凝胶材料为聚乳酸或乳酸-羟基乙酸共聚物。
3.根据权利要求1或2所述的制备方法,其特征在于,
所述人工肝脏类器官的形状为类球状体或类柱状体。
4.权利要求1-3任一项所述的制备方法得到的人工肝脏类器官作为肝脏类实验模型中的应用;所述应用包括:药物临床前检测、面向肝脏疾病的精准医疗、环境/毒性物质/污染物监测、肝脏毒理检测、肝脏疾病相关新药研发、肝脏发育生理学、肝脏病理学或药物毒性和药效试验。
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