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CN110885779A - A method for constructing a three-dimensional liver-like tissue model based on an organ chip - Google Patents

️Tue Mar 17 2020

一种基于器官芯片的三维类肝组织模型构建方法A method for constructing a three-dimensional liver-like tissue model based on an organ chip

技术领域technical field

本发明涉及微流控芯片技术和组织工程的领域，具体涉及一种基于器官芯片的三维类肝组织模型构建方法。The invention relates to the fields of microfluidic chip technology and tissue engineering, in particular to a method for constructing a three-dimensional liver-like tissue model based on an organ chip.

背景技术Background technique

肝脏是一个复杂的器官具有独特结构和有大量的基本功能包括血清清蛋白的生物合成、能量代谢、转换的药物、解毒和胆汁的生产。肝脏体外模型对疾病的研究和有效的药物测试是至关重要的。然而，由于物种间差异，动物模型尚不能真实地评估药物在人体内引起的肝毒性效应。人来源的肝体外模型可以获得更准确的候选药物信息，从而有效地弥补动物实验中的缺陷。目前主要的人源肝细胞是人原代肝细胞，具有典型的肝特异性功能，但细胞来源有限。人多能干细胞hiPSCs已被认为是一个潜在的有价值的细胞来源，可以无限供应并用来构建人肝脏体外模型，体现肝细胞特定的基本功能。一般来说hiPSCs向肝细胞分化的方法是使用一系列的化学物质或诱导因子，分化过程包括特定的内胚层诱导，肝前体细胞分化和肝细胞成熟。The liver is a complex organ with a unique structure and has a number of essential functions including serum albumin biosynthesis, energy metabolism, conversion of drugs, detoxification and bile production. Liver in vitro models are critical for disease research and effective drug testing. However, due to differences between species, animal models cannot realistically assess the hepatotoxic effects of drugs in humans. Human-derived liver in vitro models can obtain more accurate drug candidate information, thereby effectively making up for the defects in animal experiments. At present, the main human hepatocytes are primary human hepatocytes, which have typical liver-specific functions, but have limited cell sources. Human pluripotent stem cells, hiPSCs, have been recognized as a potentially valuable source of cells that can be supplied indefinitely and used to construct in vitro models of the human liver that embody hepatocyte-specific basic functions. Generally, hiPSCs differentiate into hepatocytes using a series of chemical substances or inducing factors. The differentiation process includes specific endoderm induction, hepatic precursor cell differentiation and hepatocyte maturation.

近几年来多种干细胞来源(包括成体干细胞、胚胎干细胞以及诱导多能干细胞hiPSCs)的类器官技术得到了有效发展。研究表明hiPSCs能定向分化成特异的细胞类型，并具有强大的自组装能力，能形成多种类器官，目前已有的 hiPSCs来源的类器官包括肝，肠，视网膜和大脑等。其中类肝器官的形成是由干细胞特异性分化并自组装成具有三维结构的多细胞聚集体，在体外进一步发育成具有一定结构和功能特异性的组织，一定程度上模拟了相应肝的发育过程和基本功能。人干细胞来源的类肝模型可以在模拟肝发育过程、药物测试和细胞替代疗法以及疾病模型的建立和机制研究等方面提供一个潜在的平台，有效Organoid technologies from a variety of stem cell sources, including adult stem cells, embryonic stem cells, and induced pluripotent stem cells, hiPSCs, have been effectively developed in recent years. Studies have shown that hiPSCs can be directed to differentiate into specific cell types, have strong self-assembly ability, and can form a variety of organoids. The existing organoids derived from hiPSCs include liver, intestine, retina and brain. Hepatoid organoids are formed by the specific differentiation and self-assembly of stem cells into multicellular aggregates with a three-dimensional structure, which are further developed into tissues with certain structure and function specificity in vitro, which simulates the development process of the corresponding liver to a certain extent. and basic functions. Liver-like models derived from human stem cells can provide a potential platform for simulating liver development, drug testing and cell replacement therapy, as well as disease model establishment and mechanism research.

弥补传统二维细胞培养模型以及动物模型的不足，具有广泛的应用前景。尽管这类技术有潜在的优势和应用，仍然面临很多局限和不足。首先，由于3D 细胞团的结构特点，使得内部细胞由于缺少氧气和营养，出现中心细胞坏死现象，极大限制了类肝发育的程度，包括体积大小、功能成熟度等，而在生理情况下肝组织中分布着血管网络用于提供充分的氧气和营养物质，是传统方法很难实现的；其次，实现肝组织血管化和细胞-细胞/基质间相互作用是现有方法难以达到的；另外，传统方法中如使用旋转反应器培养类器官需要消耗大量的培养基和培养空间，成本较高并且可操作性不强，不利于施加不同的条件刺激。最后，传统方法主要是利用细胞自组装和化学因子诱导形成类肝，没有时空上的因素控制及物理因素的参与。而体内肝脏发育是一个动态的过程，组织形态发生和器官形成均需考虑细胞微环境因素，如机械流体流动、生化因子、多细胞之间相互作用等等。因此，可控的细胞微环境也是实现体外3D肝模型建立需要考虑的问题。结合现有的工程化手段尤其是微流控技术，有望优化类肝技术。It makes up for the deficiencies of traditional two-dimensional cell culture models and animal models, and has broad application prospects. Despite the potential advantages and applications of this type of technology, there are still many limitations and shortcomings. First of all, due to the structural characteristics of the 3D cell clusters, the central cell necrosis occurs due to the lack of oxygen and nutrients in the internal cells, which greatly limits the degree of liver-like development, including size and functional maturity. The vascular network is distributed in the tissue to provide sufficient oxygen and nutrients, which is difficult to achieve by traditional methods; secondly, the realization of liver tissue vascularization and cell-cell/matrix interaction is difficult to achieve by existing methods; in addition, Traditional methods, such as using a rotary reactor to culture organoids, require a large amount of medium and culture space, which is costly and not easy to operate, which is not conducive to applying different conditioned stimuli. Finally, the traditional method mainly uses the self-assembly of cells and the induction of chemical factors to form liver-like cells, without the control of spatiotemporal factors and the participation of physical factors. In vivo liver development is a dynamic process, and both tissue morphogenesis and organ formation need to consider cellular microenvironmental factors, such as mechanical fluid flow, biochemical factors, and multicellular interactions. Therefore, a controllable cellular microenvironment is also an issue that needs to be considered for the establishment of an in vitro 3D liver model. Combined with existing engineering methods, especially microfluidic technology, it is expected to optimize liver-like technology.

器官芯片作为细胞培养载体，不仅可以实现通道结构尺寸的灵活设计，而且能在三维环境中模拟细胞微环境的关键因素，包括流体流动、信息交互、生化信号等等。流体控制有助于促进营养物质和氧气的交换，减少细胞凋亡或细胞团中心坏死现象，为细胞培养提供良好的生存环境。结合微流控技术不仅能解决现有设备存在体系复杂和营养物质运输欠缺的问题，还可进行细胞的实时监测和观察。但是目前将微流控技术与类肝技术相结合，优化体外类肝的形成及操作尚属空白。As a cell culture carrier, organ-on-a-chip can not only realize flexible design of channel structure size, but also simulate key factors of cell microenvironment in 3D environment, including fluid flow, information interaction, biochemical signals, etc. Fluid control helps promote the exchange of nutrients and oxygen, reduces apoptosis or necrosis in the center of the cell mass, and provides a good living environment for cell culture. Combining microfluidic technology can not only solve the problems of complex system and lack of nutrient transport in existing equipment, but also conduct real-time monitoring and observation of cells. However, it is still blank to combine microfluidic technology with liver-like technology to optimize the formation and operation of liver-like in vitro.

发明内容SUMMARY OF THE INVENTION

本发明的目的是提供一种基于器官芯片的三维类肝组织模型构建方法，该方法有效地实现了类肝的原位分化、形成和长期培养。该微流控灌流系统通过三维细胞培养和流体控制等要素，模拟体内肝形成的细胞微环境，不仅实现了类肝的原位分化、形成和长期培养，还保证了充分的营养物质和氧气的交换，并具有高通量、低成本、易操作、可原位示踪与监测的特点，为模拟肝发育、药物筛选和测试以及疾病研究提供了一个新的平台，也为其他类器官体外3D模型的建立提供一个技术支撑。The purpose of the present invention is to provide a method for constructing a three-dimensional liver-like tissue model based on an organ chip, which effectively realizes the in situ differentiation, formation and long-term culture of the liver-like tissue. The microfluidic perfusion system simulates the cellular microenvironment formed by the liver in vivo through three-dimensional cell culture and fluid control. It has the characteristics of high-throughput, low-cost, easy operation, and in situ tracking and monitoring, providing a new platform for simulating liver development, drug screening and testing, and disease research, and also for other organoids in vitro 3D The establishment of the model provides a technical support.

本发明提供了一种基于器官芯片的三维类肝组织模型构建方法，其特征在于：该模型的构建方法的主要步骤为：The invention provides a method for constructing a three-dimensional liver-like tissue model based on an organ chip, which is characterized in that: the main steps of the method for constructing the model are:

(1)器官芯片的制备与修饰；(1) Preparation and modification of organ chips;

(2)拟胚体EBs的形成；(2) Formation of embryoid body EBs;

(3)类肝在器官芯片中的分化和成熟。(3) Differentiation and maturation of hepatoids in organ-on-a-chip.

所述器官芯片主要由培养液入口、灌流通道、柱形阵列、培养液出口组成，液体由培养液入口进入后通过含有柱形阵列的灌流通道，再由出口流出。The organ chip is mainly composed of a culture medium inlet, a perfusion channel, a columnar array, and a culture medium outlet. The liquid enters from the culture medium inlet, passes through the perfusion channel containing the columnar array, and then flows out from the outlet.

所述器官芯片灌流通道宽度范围为5mm-10mm，灌流通道高度范围为1-1.3 mm，柱形阵列结构中小柱直径范围为500μm-1mm，小柱高度范围为500-800 μm，小柱间间距范围为50-100μm。The perfusion channel width of the organ chip ranges from 5 mm to 10 mm, the height of the perfusion channel ranges from 1 to 1.3 mm, the diameter of the small pillars in the columnar array structure ranges from 500 μm to 1 mm, the height of the small pillars ranges from 500 to 800 μm, and the spacing between the small pillars is in the range of 500 μm to 1 mm. The range is 50-100 μm.

所述器官芯片由上下两层不可逆封接而成，上下层材料均为透明透气的生物相容性材料聚二甲基硅氧烷的聚合物。The organ chip is formed by irreversible sealing of upper and lower layers, and the materials of the upper and lower layers are both polymers of a transparent and breathable biocompatible material polydimethylsiloxane.

本发明提供的一种器官芯片，具体制备步骤为：芯片的制备采用传统的光刻技术，将形成的SU8模板用三甲基氯硅烷蒸汽修饰，95℃烘5min，以便SU8 模板疏水尽量不粘附PDMS。之后用PDMS聚合物反模即形成上、下层PDMS 芯片。所述器官芯片层的上下两层分别经过氧气等离子体处理20-90s进行不可逆封接；封接之后，经过高温高压灭菌处理备用。An organ chip provided by the invention, the specific preparation steps are as follows: the preparation of the chip adopts traditional photolithography technology, the formed SU8 template is modified with trimethylchlorosilane vapor, and baked at 95°C for 5 minutes, so that the SU8 template is as hydrophobic as possible and not sticky With PDMS. Afterwards, the upper and lower layers of PDMS chips are formed by using the PDMS polymer reverse mold. The upper and lower layers of the organ chip layer are respectively subjected to oxygen plasma treatment for 20-90s for irreversible sealing; after sealing, they are subjected to high temperature and high pressure sterilization for use.

所述封接好的芯片用一定浓度的PF127修饰数小时，用培养基清洗数次浸泡过夜备用。The sealed chip was modified with a certain concentration of PF127 for several hours, washed with culture medium for several times and soaked overnight for use.

所述PF127修饰芯片的浓度范围为0.1％-2％，修饰时间为4-24h。The concentration range of the PF127 modified chip is 0.1%-2%, and the modification time is 4-24h.

本发明提供的EBs形成方法，具体步骤为：使用按以上步骤制备的芯片，将人多能干细胞用消化液消化成单细胞，离心500-800rpm，3-5min，用mTESR1 培养基重悬细胞至合适的细胞密度，并接种至已制备好的芯片中，细胞悬液从器官芯片培养液入口处进入，其中培养基中加入一定浓度的Y27632因子，静置培养1天。The method for forming EBs provided by the present invention includes the following specific steps: using the chip prepared according to the above steps, digesting human pluripotent stem cells into single cells with a digestive solution, centrifuging at 500-800 rpm for 3-5 min, and resuspending the cells in mTESR1 medium to Appropriate cell density, and inoculated into the prepared chip, the cell suspension enters from the entrance of the organ chip culture medium, and a certain concentration of Y27632 factor is added to the medium, and cultured for 1 day.

所述人多能干细胞为人iPSCs细胞，细胞接种密度范围为2×10³～6×10⁶ cells/ml，所述Y27632浓度范围为5-10μM。The human pluripotent stem cells are human iPSCs cells, the cell seeding density ranges from 2×10 ³ to 6×10 ⁶ cells/ml, and the Y27632 concentration ranges from 5 to 10 μM.

本发明提供的类肝在器官芯片中的分化和成熟，具体步骤为：The specific steps for the differentiation and maturation of the hepatoid in the organ chip provided by the present invention are:

(1)形成EBs 24h后开始诱导其向内胚层分化：将mTESR1培养基替换为 1640+B27培养基，其中加入高浓度的activin-A，并连续灌流5天。(1) EBs were induced to differentiate into endoderm 24h after they were formed: mTESR1 medium was replaced with 1640+B27 medium, which added high concentration of activin-A, and was perfused continuously for 5 days.

所述1640+B27培养基的基础成分为商业化的RPMI-1640培养基，需添加占总体积1％的B27；所述activin-A的浓度范围为80-120ng/ml。The basic component of the 1640+B27 medium is commercial RPMI-1640 medium, and 1% of the total volume of B27 needs to be added; the concentration range of the activin-A is 80-120 ng/ml.

(2)诱导肝前体细胞分化和增殖：在1640+B27培养基添加HGF和bFGF因子并连续灌流5天。(2) Induction of differentiation and proliferation of hepatic precursor cells: HGF and bFGF factors were added to 1640+B27 medium and perfused continuously for 5 days.

所述HGF浓度范围为20-30ng/ml，bFGF浓度范围为10-20ng/ml。The HGF concentration range was 20-30 ng/ml, and the bFGF concentration was 10-20 ng/ml.

(3)促进肝细胞进一步成熟：1640+B27培养基更换为商业化的肝细胞培养基(HCM)，另外需添加OSM因子和地塞米松(Dex)，并连续灌流5天；(3) Promote further maturation of hepatocytes: 1640+B27 medium was replaced with commercialized hepatocyte medium (HCM), and OSM factor and dexamethasone (Dex) were added, and the medium was continuously perfused for 5 days;

所述OSM浓度范围为10-20ng/ml，Dex浓度范围为10^-7-10^-6M。The OSM concentration range is 10-20ng/ml, and the Dex concentration ^range is ^10-7-10-6 M.

(4)类肝形成和长期培养：培养第15天以后，培养基去掉OSM因子，更换为只含Dex的HCM培养基，连续灌流，后续可进行长期的培养，可培养至30天并进行功能鉴定，经鉴定此方法可模拟肝的发育和形成过程。(4) Liver-like formation and long-term culture: After the 15th day of culture, the OSM factor was removed from the medium, and the medium was replaced with HCM medium containing only Dex, and the medium was continuously perfused. The subsequent long-term culture can be cultured for up to 30 days and function. It was identified that this method mimics the development and formation of the liver.

所述Dex浓度范围为10^-7-10^-6M。The Dex concentration ranged from ^10-7 to ^10-6 M.

所述器官芯片集成了灌流体系，灌流管内填充培养基，管口插入器官芯片入口处，液体流速可调节，流速为40ul/h。The organ chip integrates a perfusion system, the perfusion tube is filled with culture medium, the nozzle of the tube is inserted into the entrance of the organ chip, and the liquid flow rate can be adjusted, and the flow rate is 40ul/h.

本发明构建的一种新型的工程化的三维类肝发育模型，是一种基于器官芯片技术的可灌流的微反应器。本发明是将体内肝发育的基本原理和工程化技术结合，更好地模拟了肝形成的微环境。本发明具有成本低、操作简单、可原位追踪和实时监测等优点，可替代动物模型和传统的二维培养方式，在一定程度上模拟肝的形成与发育，为体外模拟肝形成、药物代谢、药物筛选和毒性检测等方面提供了一个强有力的技术支撑。A novel engineered three-dimensional liver-like development model constructed by the present invention is a perfusable microreactor based on organ chip technology. The invention combines the basic principle of liver development in vivo with engineering technology, and better simulates the microenvironment of liver formation. The invention has the advantages of low cost, simple operation, in-situ tracking and real-time monitoring, etc. It can replace animal models and traditional two-dimensional culture methods, simulate the formation and development of liver to a certain extent, and simulate liver formation and drug metabolism in vitro. , drug screening and toxicity testing and other aspects provide a strong technical support.

附图说明Description of drawings

图1器官芯片结构示意图，其中：1为培养液入口，2为灌流通道，3为柱形阵列，4为培养液出口。Figure 1 is a schematic diagram of the structure of the organ chip, wherein: 1 is the inlet of the culture solution, 2 is the perfusion channel, 3 is the columnar array, and 4 is the outlet of the culture solution.

图2是器官芯片上类肝分化和形成的原理图。Figure 2 is a schematic diagram of hepatoid differentiation and formation on an organ-on-a-chip.

图3是芯片上hiPSCs诱导分化成类肝的方法流程图及类肝初步表征图。其中A图为hiPSCs向类肝诱导分化的过程，主要包括内胚层诱导、肝前体细胞分化和扩增以及肝细胞成熟；B图为明场跟踪图，显示在不同时期的肝发育的形态，包含D0、D5、D10、D15、D20、D30的明场图，bars:200μm；C图为第30天分别在静态培养和灌流培养条件下的类肝组织切片的免疫荧光染色图，D图为 caspase3阳性表达的定量图，caspase3显示凋亡的细胞。Figure 3 is a flow chart of the method for inducing differentiation of hiPSCs on a chip into hepatoids and a diagram of preliminary characterization of the hepatoids. Picture A shows the process of hiPSCs differentiation into hepatoids, including endoderm induction, differentiation and expansion of hepatic precursor cells, and hepatocyte maturation; picture B shows the brightfield tracking picture, showing the morphology of liver development at different stages. Brightfield images including D0, D5, D10, D15, D20, D30, bars: 200 μm; C is the immunofluorescence staining of liver-like tissue sections under static culture and perfusion culture conditions on the 30th day, D is Quantitative plot of positive expression of caspase3, caspase3 showing apoptotic cells.

图4是比较静态和灌流培养条件下类肝在不同发育时期的组织形态学表征图；其中图A为静态和灌流条件下细胞球在第5天的内胚层基因FOXA2和 SOX17，第30天的肝细胞白蛋白ALB、细胞色素P450(CYP3A4)的相对mRNA 水平表达情况的比较；图B为静态和灌流条件下细胞球分别在第5天和第30天的SOX17、ALB、CYP3A4免疫荧光染色结果图。Figure 4 is a graph comparing the histomorphological characterization of hepatoids at different developmental stages under static and perfusion culture conditions; Figure A shows the endoderm genes FOXA2 and SOX17 of cell spheroids on day 5 under static and perfusion conditions, and the morphological characteristics of hepatoids on day 30 under static and perfusion conditions. Comparison of the relative mRNA levels of albumin ALB and cytochrome P450 (CYP3A4) in hepatocytes; Figure B shows the immunofluorescence staining results of SOX17, ALB, and CYP3A4 on cell spheroids under static and perfusion conditions on the 5th and 30th days, respectively picture.

具体实施方式Detailed ways

下面的实施例将对本发明予以进一步的说明，但并不因此而限制本发明。The following examples will further illustrate the present invention, but do not limit the present invention accordingly.

实施例1Example 1

器官芯片上hiPSCs来源的类肝的形成。Formation of hiPSCs-derived hepatoids on Organ Chips.

本发明提供了一种基于器官芯片的三维类肝组织模型构建方法，其特征在于：该模型的构建方法的主要步骤为：微流控芯片的制备与修饰、拟胚体EBs 的形成、类肝在微流控芯片中的分化和成熟。The invention provides a method for constructing a three-dimensional liver-like tissue model based on an organ chip. Differentiation and maturation in microfluidic chips.

所述器官芯片主要由培养液入口1、灌流通道2、柱形阵列3、培养液出口 4组成，液体由入口1进入后通过含有柱形阵列3的灌流通道2，再由出口4流出。该器官芯片结构示意图如图1所示。The organ chip is mainly composed of a culture fluid inlet 1, a perfusion channel 2, a columnar array 3, and a culture fluid outlet 4. The liquid enters from the inlet 1 and passes through the perfusion channel 2 containing the columnar array 3, and then flows out from the outlet 4. The schematic diagram of the organ chip structure is shown in Figure 1.

微流控芯片灌流通道宽度为8mm，灌流通道高度为1mm，柱形阵列结构中小柱直径为800μm，小柱高度为500μm，小柱间间距为50μm。The width of the perfusion channel of the microfluidic chip is 8 mm, the height of the perfusion channel is 1 mm, the diameter of the small column in the columnar array structure is 800 μm, the height of the small column is 500 μm, and the spacing between the small columns is 50 μm.

所述器官芯片由上下两层不可逆封接而成，上下层材料均为透明透气的生物相容性材料聚二甲基硅氧烷的聚合物。上下两层分别经过氧气等离子体处理 50s进行不可逆封接；封接之后，经过高温高压灭菌处理备用。The organ chip is formed by irreversible sealing of upper and lower layers, and the materials of the upper and lower layers are both polymers of a transparent and breathable biocompatible material polydimethylsiloxane. The upper and lower layers were treated with oxygen plasma for 50s for irreversible sealing; after sealing, they were sterilized by high temperature and high pressure for use.

一种器官芯片的具体必备步骤为：芯片的制备采用传统的光刻技术，将形成的SU8模板用三甲基氯硅烷蒸汽修饰，95℃烘5min，以便SU8模板疏水尽量不粘附PDMS。之后用PDMS聚合物反模即形成上、下层PDMS芯片。按权利要求书4所述封接好的芯片用一定浓度的PF127修饰数小时，培养基清洗次浸泡过夜备用。The specific necessary steps for an organ chip are as follows: the preparation of the chip adopts traditional photolithography technology, and the formed SU8 template is modified with trimethylchlorosilane vapor, and baked at 95°C for 5 minutes, so that the SU8 template is hydrophobic and does not adhere to PDMS as much as possible. Afterwards, the upper and lower layers of PDMS chips are formed by using the PDMS polymer reverse mold. According to claim 4, the sealed chip is modified with a certain concentration of PF127 for several hours, and the culture medium is washed and soaked overnight for use.

所述PF127修饰芯片的浓度为0.2％，修饰时间为24h。The concentration of the PF127 modified chip was 0.2%, and the modification time was 24h.

本发明提供的EBs形成方法，具体步骤为：使用按以上步骤制备的芯片，将人多能干细胞用消化液消化成单细胞，离心800rpm，3min，用mTESR1培养基重悬细胞至合适的细胞密度，并接种至已制备好的芯片中，细胞悬液从器官芯片培养液入口处进入，其中培养基中加入一定浓度的Y27632和bFGF因子，静置培养1天。The method for forming EBs provided by the present invention includes the following specific steps: using the chip prepared according to the above steps, digesting human pluripotent stem cells into single cells with a digestive solution, centrifuging at 800 rpm for 3 min, and resuspending the cells in mTESR1 medium to a suitable cell density , and inoculated into the prepared chip, the cell suspension enters from the entrance of the organ chip culture medium, and a certain concentration of Y27632 and bFGF factors are added to the culture medium, and cultured for 1 day.

所述人多能干细胞为人iPSCs细胞，细胞接种密度为6×10⁶cells/ml，Y27632 浓度为10μM。The human pluripotent stem cells are human iPSCs cells, the cell seeding density is 6×10 ⁶ cells/ml, and the concentration of Y27632 is 10 μM.

在以上制备的芯片中诱导类肝在器官芯片中的分化和成熟，其中微流控芯片灌流体系原理图如图2所示，诱导类肝在微流控芯片中的分化和成熟具体步骤为：In the chip prepared above, the differentiation and maturation of hepatoids in the organ chip is induced. The schematic diagram of the microfluidic chip perfusion system is shown in Figure 2. The specific steps for inducing the differentiation and maturation of hepatoids in the microfluidic chip are as follows:

(1)形成EBs一天后开始向内胚层诱导分化：将mTESR1培养基替换为 1640+B27培养基，其中加入高浓度的activin-A，并连续灌流5天。(1) Induction of differentiation into endoderm started one day after the formation of EBs: mTESR1 medium was replaced with 1640+B27 medium, which added high concentration of activin-A, and was continuously perfused for 5 days.

所述1640+B27培养基的基础成分为商业化的RPMI-1640培养基，需添加占总体积1％的B27；The basic component of the 1640+B27 medium is the commercialized RPMI-1640 medium, and 1% of the total volume of B27 needs to be added;

所述activin-A的浓度为100ng/ml。The concentration of the activin-A was 100 ng/ml.

(2)诱导肝前体细胞分化和增殖：在1640+B27培养基添加HGF和bFGF因子并连续灌流5天。所述HGF浓度为20ng/ml，bFGF浓度为10ng/ml。(2) Induction of differentiation and proliferation of hepatic precursor cells: HGF and bFGF factors were added to 1640+B27 medium and perfused continuously for 5 days. The HGF concentration was 20 ng/ml and the bFGF concentration was 10 ng/ml.

所述OSM浓度为10ng/ml，Dex浓度为10^-7M。(4)类肝的长期培养：培养第 15天以后，培养基去掉OSM因子，更换为只含Dex的HCM培养基，连续灌流，后续可进行长期的培养，可培养至30天并进行功能鉴定，经鉴定此方法可模拟肝的发育和形成过程。所述Dex浓度为10^- ⁷M。所述器官芯片集成了灌流体系，灌流管内填充培养基，管口插入器官芯片入口处，液体流速可调节，流速为40 ul/h。按以上步骤诱导肝分化相关的原理图及初步表征图如图3所示，说明hiPSCs 在微流控灌流体系下可成功发育成类肝。The OSM concentration was 10 ng/ml and the Dex concentration was 10 ^-7 M. (4) Long-term culture of hepatoids: After the 15th day of culture, the OSM factor was removed from the medium, and the medium was replaced with HCM medium containing only Dex, with continuous perfusion, followed by long-term culture, which can be cultured for up to 30 days and functional identification. , which was identified as mimicking the development and formation of the liver. The Dex concentration was ^{10 −7} ^M. The organ chip integrates a perfusion system, the perfusion tube is filled with culture medium, the nozzle of the tube is inserted into the entrance of the organ chip, and the liquid flow rate is adjustable, and the flow rate is 40 ul/h. The schematic diagram and preliminary characterization diagram related to the induction of liver differentiation according to the above steps are shown in Figure 3, indicating that hiPSCs can successfully develop into liver-like cells under the microfluidic perfusion system.

实施例2Example 2

类肝形成过程中肝相关蛋白的表达。Expression of liver-associated proteins during hepatoid formation.

取器官芯片上分别在静态和灌流培养条件下诱导到第5天和第30天的类肝细胞球进行real-time PCR表征，如图4A所示，结果表明在器官芯片灌流培养条件下内胚层基因(SOX17,FOXA2)和肝相关基因(ALB,CYP3A4)的表达明显高于静态培养条件下相关基因的表达。结果表明类肝在微流控灌流体系下可进行良好的内胚层分化和进一步的肝发育。The hepatocyte-like spheroids induced to the 5th and 30th day under static and perfusion culture conditions on the Organ Chip, respectively, were characterized by real-time PCR, as shown in Figure 4A. The expressions of genes (SOX17, FOXA2) and liver-related genes (ALB, CYP3A4) were significantly higher than those of related genes under static culture conditions. The results indicated that the liver-like cells could undergo good endoderm differentiation and further liver development under the microfluidic perfusion system.

取器官芯片上分别在静态和灌流培养条件下诱导到5天和第30天的细胞球分别进行冷冻切片，方法如下：4％多聚甲醛进行细胞固定20min，PBS缓冲液冲洗三次，每次10min；30％蔗糖4℃过夜脱水；OCT包埋，室温存放30min， 80℃凝固；冷冻切片，厚度为10-20μm，并将其贴附在静电吸附的载玻片上。然后进行免疫荧光染色，方法为：将带有切片的载玻片置于PBS缓冲液中浸泡 5min；0.1％triton X-100致孔剂作用10min，PBS缓冲液冲洗1次，5min；山羊封闭血清室温作用1h；一抗(SOX17、ALB、CYP3A4)1:500稀释，4℃过夜孵育，PBS缓冲液冲洗3次。二抗(Fluorescence 488/594标记的山羊抗兔或鼠 IgG以及Cy3标记的驴抗羊IgG)1:200稀释，室温孵育1h，PBS缓冲液冲洗3 次；冲洗完毕后加入1:4000稀释的DAPI工作液，荧光显微镜下拍照，记录相应蛋白的表达情况，结果如图4B所示，Scale bars：50μm。在器官芯片灌流培养条件下。结果表明在器官芯片灌流培养条件下内胚层标志物SOX17和肝相关标志物ALB和CYP3A4的蛋白表达明显高于静态培养条件下相关蛋白的表达。结果表明类肝在微流控灌流体系下可进行良好的内胚层分化和进一步的肝发育，与real-timePCR结果一致。Cell spheroids induced to 5 days and 30 days under static and perfusion culture conditions on the organ chip were taken for cryosectioning, respectively. The method was as follows: 4% paraformaldehyde was used for cell fixation for 20 min, and PBS buffer was washed three times for 10 min each time. 30% sucrose at 4°C overnight dehydration; OCT embedded, stored at room temperature for 30 min, and solidified at 80°C; frozen section, 10-20 μm thick, and attached to an electrostatically adsorbed glass slide. Then immunofluorescence staining was carried out, the method was as follows: the slides with sections were soaked in PBS buffer for 5 minutes; 0.1% triton X-100 porogen was used for 10 minutes, washed once with PBS buffer for 5 minutes; goat blocking serum Act at room temperature for 1 h; primary antibodies (SOX17, ALB, CYP3A4) were diluted 1:500, incubated at 4°C overnight, and washed three times with PBS buffer. Secondary antibodies (Fluorescence 488/594-labeled goat anti-rabbit or mouse IgG and Cy3-labeled donkey anti-goat IgG) were diluted at 1:200, incubated at room temperature for 1 h, washed three times with PBS buffer; after washing, 1:4000 diluted DAPI was added The working solution was photographed under a fluorescence microscope to record the expression of the corresponding protein. The results are shown in Figure 4B, Scale bars: 50 μm. Organ-on-a-chip perfusion culture conditions. The results showed that the protein expression of endoderm marker SOX17 and liver-related markers ALB and CYP3A4 under the perfusion culture condition of organ chip was significantly higher than that under static culture condition. The results showed that the liver-like cells could undergo good endoderm differentiation and further liver development under the microfluidic perfusion system, which was consistent with the real-time PCR results.

实施例3Example 3

器官芯片上hiPSCs来源的类肝的形成。Formation of hiPSCs-derived hepatoids on Organ Chips.

所述器官芯片主要由培养液入口1、灌流通道2、柱形阵列3、培养液出口 4组成，液体由入口1进入后通过含有柱形阵列3的灌流通道2，再由出口4流出。该器官芯片结构示意图如图1所示，微流控芯片灌流通道宽度为5mm，灌流通道高度为1.3mm，柱形阵列结构中小柱直径为500μm，小柱高度为800μm，小柱间间距为100μm。The organ chip is mainly composed of a culture fluid inlet 1, a perfusion channel 2, a columnar array 3, and a culture fluid outlet 4. The liquid enters from the inlet 1 and passes through the perfusion channel 2 containing the columnar array 3, and then flows out from the outlet 4. The schematic diagram of the organ chip structure is shown in Figure 1. The width of the perfusion channel of the microfluidic chip is 5 mm, the height of the perfusion channel is 1.3 mm, the diameter of the small column in the columnar array structure is 500 μm, the height of the small column is 800 μm, and the spacing between the small columns is 100 μm. .

所述器官芯片由上下两层不可逆封接而成，上下层材料均为透明透气的生物相容性材料聚二甲基硅氧烷的聚合物。上下两层分别经过氧气等离子体处理 90s进行不可逆封接；封接之后，经过高温高压灭菌处理备用。The organ chip is formed by irreversible sealing of upper and lower layers, and the materials of the upper and lower layers are both polymers of a transparent and breathable biocompatible material polydimethylsiloxane. The upper and lower layers were treated with oxygen plasma for 90s for irreversible sealing; after sealing, they were sterilized by high temperature and high pressure for use.

所述PF127修饰芯片的浓度为2％，修饰时间为4h。The concentration of the PF127 modified chip was 2%, and the modification time was 4h.

所述人多能干细胞为人iPSCs细胞，细胞接种密度为4×10³cells/ml，Y27632 浓度为5μM。The human pluripotent stem cells are human iPSCs cells, the cell seeding density is 4×10 ³ cells/ml, and the concentration of Y27632 is 5 μM.

所述activin-A的浓度为120ng/ml。The concentration of the activin-A was 120ng/ml.

(2)诱导肝前体细胞分化和增殖：在1640+B27培养基添加HGF和bFGF因子并连续灌流5天。所述HGF浓度为30ng/ml，bFGF浓度为20ng/ml。(2) Induction of differentiation and proliferation of hepatic precursor cells: HGF and bFGF factors were added to 1640+B27 medium and perfused continuously for 5 days. The HGF concentration was 30 ng/ml and the bFGF concentration was 20 ng/ml.

所述OSM浓度为20ng/ml，Dex浓度为10^-6M。The OSM concentration was 20 ng/ml and the Dex concentration was 10 ^-6 M.

(4)类肝的长期培养：培养第15天以后，培养基去掉OSM因子，更换为只含 Dex的HCM培养基，连续灌流，后续可进行长期的培养，可培养至30天并进行功能鉴定，经鉴定此方法可模拟肝的发育和形成过程。所述Dex浓度为10^-6M。所述器官芯片集成了灌流体系，灌流管内填充培养基，管口插入器官芯片入口处，液体流速可调节，流速为25ul/h。按以上步骤诱导肝分化相关的原理图及初步表征图与图3类似，说明hiPSCs在微流控灌流体系下可成功发育成类肝。(4) Long-term culture of hepatoids: After the 15th day of culture, the OSM factor was removed from the medium, and the medium was replaced with HCM medium containing only Dex, with continuous perfusion, followed by long-term culture, which can be cultured for up to 30 days and functional identification. , which was identified as mimicking the development and formation of the liver. The Dex concentration was 10 ^-6 M. The organ chip integrates a perfusion system, the perfusion tube is filled with culture medium, the nozzle is inserted into the entrance of the organ chip, and the liquid flow rate is adjustable, and the flow rate is 25ul/h. The schematic diagram and preliminary characterization diagram related to the induction of liver differentiation according to the above steps are similar to those in Figure 3, indicating that hiPSCs can successfully develop into liver-like cells under the microfluidic perfusion system.

实施例4Example 4

器官芯片上hiPSCs来源的类肝的形成。Formation of hiPSCs-derived hepatoids on Organ Chips.

所述器官芯片主要由培养液入口1、灌流通道2、柱形阵列3、培养液出口 4组成，液体由入口1进入后通过含有柱形阵列3的灌流通道2，再由出口4流出。该器官芯片结构示意图如图1所示，微流控芯片灌流通道宽度为10mm，灌流通道高度为1mm，柱形阵列结构中小柱直径为900μm，小柱高度为800μm，小柱间间距为100μm。The organ chip is mainly composed of a culture fluid inlet 1, a perfusion channel 2, a columnar array 3, and a culture fluid outlet 4. The liquid enters from the inlet 1 and passes through the perfusion channel 2 containing the columnar array 3, and then flows out from the outlet 4. The schematic diagram of the organ chip structure is shown in Figure 1. The width of the perfusion channel of the microfluidic chip is 10mm, the height of the perfusion channel is 1mm, the diameter of the small column in the columnar array structure is 900 μm, the height of the small column is 800 μm, and the spacing between the small columns is 100 μm.

所述器官芯片由上下两层不可逆封接而成，上下层材料均为透明透气的生物相容性材料聚二甲基硅氧烷的聚合物。上下两层分别经过氧气等离子体处理 20s进行不可逆封接；封接之后，经过高温高压灭菌处理备用。The organ chip is formed by irreversible sealing of upper and lower layers, and the materials of the upper and lower layers are both polymers of a transparent and breathable biocompatible material polydimethylsiloxane. The upper and lower layers were treated with oxygen plasma for 20s for irreversible sealing; after sealing, they were sterilized by high temperature and high pressure for use.

所述PF127修饰芯片的浓度为0.1％，修饰时间为24h。The concentration of the PF127 modified chip was 0.1%, and the modification time was 24h.

所述人多能干细胞为人iPSCs细胞，细胞接种密度为6×10⁶cells/ml，Y27632 浓度为8μM。The human pluripotent stem cells are human iPSCs cells, the cell seeding density is 6×10 ⁶ cells/ml, and the concentration of Y27632 is 8 μM.

所述activin-A的浓度为80ng/ml。The concentration of the activin-A was 80 ng/ml.

(2)诱导肝前体细胞分化和增殖：在1640+B27培养基添加HGF和bFGF因子并连续灌流5天。所述HGF浓度为25ng/ml，bFGF浓度为15ng/ml。(2) Induction of differentiation and proliferation of hepatic precursor cells: HGF and bFGF factors were added to 1640+B27 medium and perfused continuously for 5 days. The HGF concentration was 25ng/ml and the bFGF concentration was 15ng/ml.

所述OSM浓度为15ng/ml，Dex浓度为10^-7M。The OSM concentration was 15 ng/ml and the Dex concentration was 10 ^-7 M.

(4)类肝的长期培养：培养第15天以后，培养基去掉OSM因子，更换为只含 Dex的HCM培养基，连续灌流，后续可进行长期的培养，可培养至30天并进行功能鉴定，经鉴定此方法可模拟肝的发育和形成过程。所述Dex浓度为10^-7M。所述器官芯片集成了灌流体系，灌流管内填充培养基，管口插入器官芯片入口处，液体流速可调节，流速为60ul/h。按以上步骤诱导肝分化相关的原理图及初步表征图与图3类似，说明hiPSCs在微流控灌流体系下可成功发育成类肝。(4) Long-term culture of hepatoids: After the 15th day of culture, the OSM factor was removed from the medium, and the medium was replaced with HCM medium containing only Dex, with continuous perfusion, followed by long-term culture, which can be cultured for up to 30 days and functional identification. , which was identified as mimicking the development and formation of the liver. The Dex concentration was 10 ^-7 M. The organ chip integrates a perfusion system, the perfusion tube is filled with culture medium, the nozzle of the tube is inserted into the entrance of the organ chip, and the liquid flow rate can be adjusted, and the flow rate is 60ul/h. The schematic diagram and preliminary characterization diagram related to the induction of liver differentiation according to the above steps are similar to those in Figure 3, indicating that hiPSCs can successfully develop into liver-like cells under the microfluidic perfusion system.