CN117586876A - A vascularized organoid chip and vascularized organoid - Google Patents

A vascularized organoid chip and vascularized organoid Download PDF

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CN117586876A
CN117586876A CN202311288041.0A CN202311288041A CN117586876A CN 117586876 A CN117586876 A CN 117586876A CN 202311288041 A CN202311288041 A CN 202311288041A CN 117586876 A CN117586876 A CN 117586876A
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田甜
王思思
赵蓉
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Danwang Medical Technology (Hangzhou) Co.,Ltd.
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Abstract

The invention provides a vascularized organoid chip and a vascularized organoid, wherein the vascularized organoid chip can enlarge the contact surface of cells and a culture medium based on a fluid limiting structure, and promote the spontaneous generation of a vascular network through fluid stimulation, so that a vascularized organoid model is obtained. The invention provides a feasible method for constructing the organoid vascularization in-vitro model, simulates a more bionic in-vivo microenvironment, and simultaneously the obtained model can be used for research of immune co-culture, angiogenesis and the like.

Description

一种血管化类器官芯片及血管化类器官A vascularized organoid chip and vascularized organoid

技术领域Technical field

本发明涉及类器官培养技术领域，尤其涉及一种血管化类器官芯片及血管化类器官。The present invention relates to the technical field of organoid culture, and in particular to a vascularized organoid chip and vascularized organoids.

背景技术Background technique

类器官由于其具备的自我更新和自组织能力，并表现出与起源组织相似的器官功能，能更好地用于模拟器官组织的发生过程及生理病理状态，因而在基础研究以及临床诊疗方面具有广阔的应用前景。从2009年荷兰科学家Hans Clevers实验室培育出首个肠道类器官，经过十余年的发展，类器官现今已作为一种优质的体外模型用于临床前癌症的治疗检测及药物药效和毒性测试，并逐渐走向肿瘤癌症基础研究和临床治疗的中央舞台，此外，不少实验室及企业致力于肿瘤类类器官生物库的建立使生理学相关的药物筛选成为可能。然而，目前的类器官培养相较于人体正常组织器官的生理环境由于缺乏血管结构而无法长期培养，在类器官体积增长至一定大小时会出现因培养基及氧气，营养物质等无法进入而导致的类器官中心细胞坏死现象。此外，血管化使类器官模型更能模拟体内微环境，与免疫细胞共培养可模拟体内免疫微环境。Due to their ability to self-renew and self-organize, and to exhibit similar organ functions to the tissue of origin, organoids can better be used to simulate the occurrence process and physiological and pathological conditions of organ tissues, so they have great potential in basic research and clinical diagnosis and treatment. Broad application prospects. The first intestinal organoid was cultivated in the laboratory of Dutch scientist Hans Clevers in 2009. After more than ten years of development, organoids are now used as a high-quality in vitro model for preclinical cancer treatment testing and drug efficacy and toxicity. Testing, and gradually moving towards the central stage of basic research and clinical treatment of tumors and cancers. In addition, many laboratories and companies are committed to the establishment of tumor organoid biobanks to make physiologically relevant drug screening possible. However, compared with the physiological environment of normal human tissues and organs, the current organoid culture cannot be cultured for a long time due to the lack of vascular structure. When the volume of organoids grows to a certain size, problems may occur due to the inability of the culture medium, oxygen, nutrients, etc. to enter. The phenomenon of cell necrosis in the center of organoids. In addition, vascularization enables organoid models to better simulate the in vivo microenvironment, and co-culture with immune cells can simulate the in vivo immune microenvironment.

利用生物3D打印技术及微流控芯片等技术可实现体外血管系统的重建，目前已有不少研究者基于此两项技术实现基于水凝胶的体外三维血管网络构建。但3D打印技术构建的血管网络主要由人为设计，与人体本身血管网络存在一定的差别。基于微流控芯片的三维血管网络由细胞自发形成，与体内血管具有形态结构的高度相似性，但是现有的芯片基本采用微柱结构限制凝胶的空间分布，此结构由于微柱结构本身会占据一部分空间，导致细胞与培养基的接触面积减少，此外，微柱结构也可能挡住自发形成的血管结构端口，影响血管灌注。同时现有的微流控血管化芯片基本为封闭式结构，且流道间利用微柱隔开，不利于培养基的流通，极易造成非均匀或非充分的流体刺激。此外，芯片基本为封闭结构，往往只能依赖于人工操作且操作难度较大，同时芯片中培养的器官组织难以取出，限制了后续的分析检测手段。此种封闭芯片也无法与自动化加液等仪器设备适配，不满足自动化和通量化的需求。现有开放式血管化器官芯片采用PMMA和玻璃通过双面胶粘合的方式制作血管芯片，芯片制作完成后在PMMA与玻璃接触外侧再涂抹PDMS固化后增加密封性。水凝胶用枪头通过储液槽一侧打入圆环结构底部，通过毛细力使水凝胶分布于中间的圆环结构底部，形成环状。水凝胶的厚度由圆环结构与基底间的空隙高度决定，水凝胶的宽度由圆环结构的宽度决定。但该方法存在以下问题：1，需要使用切割机对双面胶进行相应形状切割后与上层PMMA对准粘合，操作过程较繁琐，且对准过程中易产生误差造成芯片结构的细微差别；2，由于水凝胶加入时需倾斜枪头注入，自动化仪器难以满足其需求，不利于通量化；3，后续与细胞球进行共培养时细胞球从中间孔中加入，由于距离原因导致血管很难进入细胞球内部得到血管化细胞微球；4，血管结构位于圆环结构底部，与封闭式芯片一样存在样品取出困难，无法满足后续多样性分析检测手段的需求。The reconstruction of the vascular system in vitro can be achieved using technologies such as bio-3D printing technology and microfluidic chips. Currently, many researchers have used these two technologies to construct three-dimensional vascular networks in vitro based on hydrogels. However, the vascular network constructed by 3D printing technology is mainly designed by humans and is somewhat different from the human body's own vascular network. The three-dimensional vascular network based on microfluidic chips is formed spontaneously by cells and is highly similar in morphological structure to blood vessels in the body. However, existing chips basically use micro-pillar structures to limit the spatial distribution of gels. This structure will cause problems due to the micro-pillar structure itself. Occupying part of the space, the contact area between cells and culture medium is reduced. In addition, the micro-column structure may also block the ports of spontaneously formed vascular structures, affecting vascular perfusion. At the same time, the existing microfluidic vascularization chip is basically a closed structure, and the flow channels are separated by micro-pillars, which is not conducive to the circulation of the culture medium and can easily cause non-uniform or insufficient fluid stimulation. In addition, the chip is basically a closed structure, which often relies on manual operation and is difficult to operate. At the same time, the organs and tissues cultured in the chip are difficult to remove, which limits subsequent analysis and detection methods. This kind of closed chip cannot be adapted to instruments and equipment such as automated liquid addition, and does not meet the needs of automation and throughput. The existing open vascularized organ chip uses PMMA and glass to be bonded with double-sided tape to make the vascular chip. After the chip is made, PDMS is applied to the outside of the contact between PMMA and glass to increase the sealing. Use a pipette tip to drive the hydrogel into the bottom of the ring structure through one side of the liquid reservoir. The hydrogel is distributed at the bottom of the ring structure in the middle through capillary force to form a ring shape. The thickness of the hydrogel is determined by the height of the gap between the ring structure and the substrate, and the width of the hydrogel is determined by the width of the ring structure. However, this method has the following problems: 1. It is necessary to use a cutting machine to cut the double-sided tape into corresponding shapes and then align and bond it with the upper layer PMMA. The operation process is cumbersome, and errors are prone to occur during the alignment process, resulting in subtle differences in the chip structure; 2. Since the pipette tip needs to be tilted when adding hydrogel, automated instruments are difficult to meet its needs, which is not conducive to throughput; 3. When co-culturing with cell spheroids, the cell spheroids are added from the middle hole, and the blood vessels are very small due to the distance. It is difficult to enter the interior of the cell sphere to obtain vascularized cell microspheres; 4. The blood vessel structure is located at the bottom of the ring structure. Like the closed chip, there are difficulties in sample removal, and it cannot meet the needs of subsequent diversity analysis and detection methods.

因此，亟需提供一种血管化类器官芯片以解决上述技术问题。Therefore, there is an urgent need to provide a vascularized organoid chip to solve the above technical problems.

发明内容Contents of the invention

本发明旨在至少一定程度上解决相关技术中的技术问题之一。为此，本发明提供了一种血管化类器官芯片及血管化类器官，所述血管化类器官芯片基于流体限位结构可增大细胞与培养基的接触面，通过流体刺激促进血管网络自发生成，进而得到血管化类器官模型。本发明为类器官血管化体外模型构建提供了可行方法，模拟了更仿生的体内微环境，同时得到的模型也可用于免疫共培养、血管新生等研究。The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, the present invention provides a vascularized organoid chip and vascularized organoids. The vascularized organoid chip is based on a fluid limiting structure that can increase the contact surface between cells and culture medium and promote the spontaneous activation of the vascular network through fluid stimulation. Generate, and then obtain a vascularized organoid model. The present invention provides a feasible method for constructing an in vitro model of organoid vascularization, simulating a more bionic in vivo microenvironment, and the obtained model can also be used for immune co-culture, angiogenesis and other research.

为此，本发明第一方面提供了一种血管化类器官芯片，所述血管化类器官芯片包括依次设置的储液层、细胞培养层和基底层；To this end, the first aspect of the present invention provides a vascularized organoid chip, which includes a liquid storage layer, a cell culture layer and a basal layer arranged in sequence;

其中，所述储液层设置至少一个通孔单元，每个所述通孔单元包括依次排列的第一通孔、第二通孔和第三通孔，相邻通孔的直径呈相异设置；Wherein, the liquid storage layer is provided with at least one through hole unit, each of the through hole units includes a first through hole, a second through hole and a third through hole arranged in sequence, and the diameters of adjacent through holes are arranged differently. ;

所述细胞培养层设置至少一个培养单元，所述培养单元与所述通孔单元一一对应，每个所述培养单元包括依次排列的第一微孔、第二微孔和第三微孔，所述第一微孔、第二微孔和第三微孔分别与所述第一通孔、第二通孔和第三通孔一一对应，所述第二微孔底部形成目标高度的间隙以形成通道，所述第一微孔、第二微孔和第三微孔通过所述通道实现联通。The cell culture layer is provided with at least one culture unit, the culture unit corresponds to the through-hole unit one-to-one, and each culture unit includes first micropores, second micropores and third micropores arranged in sequence, The first microhole, the second microhole and the third microhole correspond to the first through hole, the second through hole and the third through hole respectively, and the bottom of the second microhole forms a gap of a target height. To form a channel, the first micropore, the second micropore and the third micropore are connected through the channel.

本发明提供了一种基于流体限位结构的血管化类器官芯片，该结构可增大细胞与培养基的接触面，通过流体刺激促进血管网络自发生成，得到血管化类器官芯片模型。该血管化类器官芯片解决了以下问题：The present invention provides a vascularized organoid chip based on a fluid limiting structure, which can increase the contact surface between cells and culture medium, promote the spontaneous generation of vascular networks through fluid stimulation, and obtain a vascularized organoid chip model. This vascularized organoid chip solves the following problems:

1)解决了目前芯片大多采用微柱阵列等微结构来控制水凝胶空间分布，进而造成非均匀或非充分的流体刺激问题；2)解决了现有封闭式芯片样品难以取出以及后续分析手段受限的问题；3)解决了封闭式芯片只能手动操作难以匹配自动化加样设备的问题，4)本芯片结构设计避免了由于对准键合中的误差而造成的芯片结构性差异。1) It solves the problem that most current chips use microstructures such as micropillar arrays to control the spatial distribution of hydrogels, thereby causing non-uniform or insufficient fluid stimulation; 2) It solves the problem of difficulty in removing samples from existing closed chips and subsequent analysis methods. Limited problems; 3) It solves the problem that closed chips can only be operated manually and are difficult to match automated sampling equipment. 4) This chip structure design avoids chip structural differences caused by errors in alignment bonding.

根据本发明的实施例，所述储液层、细胞培养层和基底层的厚度比为(20-50):2:1。According to an embodiment of the present invention, the thickness ratio of the liquid reservoir layer, cell culture layer and basal layer is (20-50):2:1.

根据本发明的实施例，所述储液层的厚度为5-10mm。由此满足培养基的需求量。According to an embodiment of the present invention, the thickness of the liquid storage layer is 5-10 mm. This meets the demand for culture media.

根据本发明的实施例，所述细胞培养层的厚度为0.2-1mm。由此便于水凝胶材料的灌注，避免出现凝胶材料粘附于孔壁而无法铺满孔底部的情况。According to an embodiment of the present invention, the thickness of the cell culture layer is 0.2-1 mm. This facilitates the pouring of the hydrogel material and avoids the situation where the gel material adheres to the hole wall and cannot cover the bottom of the hole.

根据本发明的实施例，所述基底层的厚度为0.1-0.5mm。由此便于后续通过显微镜观察。According to an embodiment of the present invention, the thickness of the base layer is 0.1-0.5 mm. This facilitates subsequent observation through a microscope.

根据本发明的实施例，所述细胞培养层的厚度与所述目标高度的比为(1.2-1.5):1。由此满足第二微孔中的血管化细胞和/或类器官细胞与凝胶混合物不会溢出至两侧流道中。According to an embodiment of the present invention, the ratio of the thickness of the cell culture layer to the target height is (1.2-1.5):1. This ensures that the mixture of vascularized cells and/or organoid cells and gel in the second micropore will not overflow into the flow channels on both sides.

根据本发明的实施例，所述目标高度为200-500μm。According to an embodiment of the present invention, the target height is 200-500 μm.

根据本发明的实施例，所述第二微孔的周缘底部具有楔形结构以形成目标高度的间隙，所述楔形结构的宽度沿着所述储液层到所述基底层方向逐渐缩小。According to an embodiment of the present invention, the peripheral bottom of the second micropore has a wedge-shaped structure to form a gap of a target height, and the width of the wedge-shaped structure gradually decreases along the direction from the liquid storage layer to the base layer.

根据本发明的实施例，所述第一通孔和/或所述第三通孔的直径大于所述第二通孔。由此满足培养基的需求量。According to an embodiment of the present invention, the diameter of the first through hole and/or the third through hole is larger than that of the second through hole. This meets the demand for culture media.

根据本发明的实施例，所述第一通孔和所述第三通孔的直径相同。According to an embodiment of the present invention, the first through hole and the third through hole have the same diameter.

根据本发明的实施例，所述第二微孔的直径小于所述第二通孔的直径。由此使得血管化细胞和/或类器官细胞与水凝胶混合物集中在第二微孔中，避免由于表面张力的影响，在添加血管化细胞和/或类器官细胞与水凝胶混合物时出现凝胶黏附在孔壁上无法填充整个微孔。同时也减少了水凝胶材料的使用量，节约成本。According to an embodiment of the present invention, a diameter of the second micropore is smaller than a diameter of the second through hole. This allows the mixture of vascularized cells and/or organoid cells and hydrogel to be concentrated in the second micropore, avoiding the effect of surface tension when adding the mixture of vascularized cells and/or organoid cells and hydrogel. The gel adheres to the pore walls and cannot fill the entire micropore. At the same time, it also reduces the use of hydrogel materials and saves costs.

根据本发明的实施例，所述第一通孔、第二通孔、第三通孔的直径比为8:5:8。According to an embodiment of the present invention, the diameter ratio of the first through hole, the second through hole, and the third through hole is 8:5:8.

根据本发明的实施例，所述第一通孔的直径为8mm，所述第二通孔的直径为5mm，所述第三通孔的直径为8mm。According to an embodiment of the present invention, the diameter of the first through hole is 8 mm, the diameter of the second through hole is 5 mm, and the diameter of the third through hole is 8 mm.

根据本发明的实施例，所述第二微孔的直径为2mm。According to an embodiment of the present invention, the diameter of the second micropore is 2 mm.

根据本发明的实施例，所述细胞培养层与所述基底层相接触。According to an embodiment of the invention, the cell culture layer is in contact with the basal layer.

根据本发明的实施例，所述储液层的材质为PMMA、PS、COC中的一种或多种。According to an embodiment of the present invention, the material of the liquid storage layer is one or more of PMMA, PS, and COC.

根据本发明的实施例，所述细胞培养层的材质为PMMA、PS、PC中的一种或多种。According to an embodiment of the present invention, the material of the cell culture layer is one or more of PMMA, PS, and PC.

根据本发明的实施例，所述基底层的材质为玻璃、PC、PS中的一种或多种。According to an embodiment of the present invention, the material of the base layer is one or more of glass, PC, and PS.

本发明第二方面提供了第一方面所述的血管化类器官芯片在制备血管模型或血管化类器官中的应用。A second aspect of the present invention provides the use of the vascularized organoid chip described in the first aspect in preparing blood vessel models or vascularized organoids.

本发明第三方面提供了一种血管模型的制备方法，所述制备方法采用第一方面所述的血管化类器官芯片，包括将血管化细胞与水凝胶材料混合后注入所述第二通孔，向所述第一通孔与第三通孔中的至少一个和所述第二通孔注入培养基，进行培养。A third aspect of the present invention provides a method for preparing a blood vessel model. The preparation method uses the vascularized organoid chip described in the first aspect, including mixing vascularized cells with a hydrogel material and then injecting it into the second channel. hole, inject culture medium into at least one of the first through hole, the third through hole and the second through hole for culturing.

根据本发明的实施例，所述血管化细胞包括人脐静脉内皮细胞、人肺成纤维细胞。According to an embodiment of the present invention, the vascularized cells include human umbilical vein endothelial cells and human lung fibroblasts.

根据本发明的实施例，所述水凝胶材料包括选自明胶或基质胶。According to an embodiment of the present invention, the hydrogel material includes gelatin or Matrigel.

根据本发明的实施例，所述制备方法还包括将所述血管化类器官芯片置于流体剪切力细胞培养自动化装置或半自动化装置。According to an embodiment of the present invention, the preparation method further includes placing the vascularized organoid chip in a fluid shear force cell culture automated device or semi-automated device.

本发明第四方面提供了一种血管化类器官的制备方法，所述制备方法采用第一方面所述的血管化类器官芯片，包括将血管化细胞、类器官与水凝胶材料混合后注入所述第二通孔，向所述第一通孔与第三通孔中的至少一个和所述第二通孔注入培养基，进行培养。A fourth aspect of the present invention provides a method for preparing vascularized organoids. The preparation method uses the vascularized organoid chip described in the first aspect, including mixing vascularized cells, organoids and hydrogel materials and then injecting The second through hole injects culture medium into at least one of the first through hole and the third through hole and the second through hole for culture.

根据本发明的实施例，所述血管化细胞包括人脐静脉内皮细胞、人肺成纤维细胞。According to an embodiment of the present invention, the vascularized cells include human umbilical vein endothelial cells and human lung fibroblasts.

根据本发明的实施例，所述类器官包括选自肿瘤类器官、胰腺类器官、肝脏类器官、肾脏类器官、胃类器官、肠类器官中的至少一种。According to an embodiment of the present invention, the organoids include at least one selected from the group consisting of tumor organoids, pancreatic organoids, liver organoids, kidney organoids, gastric organoids, and intestinal organoids.

根据本发明的实施例，所述肿瘤类器官包括选自肠癌类器官、肺癌类器官、胃癌类器官、胰腺癌类器官中的至少一种。According to an embodiment of the present invention, the tumor organoids include at least one selected from the group consisting of intestinal cancer organoids, lung cancer organoids, gastric cancer organoids, and pancreatic cancer organoids.

根据本发明的实施例，所述类器官包括选自肠癌类器官。According to an embodiment of the invention, the organoids comprise intestinal cancer organoids.

根据本发明的实施例，所述水凝胶材料包括选自明胶或基质胶。According to an embodiment of the present invention, the hydrogel material includes gelatin or Matrigel.

根据本发明的实施例，所述方法还包括将所述血管化类器官芯片置于流体剪切力细胞培养自动化装置或半自动化装置。According to an embodiment of the present invention, the method further includes placing the vascularized organoid chip in a fluid shear force cell culture automated device or semi-automated device.

本发明第五方面提供了一种血管模型，所述血管模型通过第三方面所述的制备方法得到。A fifth aspect of the present invention provides a blood vessel model, which is obtained by the preparation method described in the third aspect.

本发明第六方面提供了一种血管化类器官，所述血管化类器官通过第四方面所述的制备方法得到。A sixth aspect of the present invention provides a vascularized organoid, which is obtained by the preparation method described in the fourth aspect.

本发明第七方面提供了第五方面所述的血管模型或第六方面所述的血管化类器官在药物筛选中的应用。A seventh aspect of the present invention provides the application of the blood vessel model described in the fifth aspect or the vascularized organoid described in the sixth aspect in drug screening.

本发明相对于现有技术的有益效果：The beneficial effects of the present invention compared with the prior art:

1、本发明提供的血管化类器官芯片底部具有目标高度的流道，可防止中间通孔血管化细胞和/或类器官细胞不会溢出至两侧流道中，此结构与传统的微柱型设计相比能够大大增加细胞与培养基的接触面积，进而实现更加均匀及充分的流体刺激；1. The bottom of the vascularized organoid chip provided by the present invention has a flow channel with a target height, which can prevent the vascularized cells and/or organoid cells from overflowing into the flow channels on both sides of the middle through hole. This structure is different from the traditional micro-column type. The design can greatly increase the contact area between cells and culture medium, thereby achieving more uniform and sufficient fluid stimulation;

2、本发明提供的血管化类器官芯片上层储液层较厚可满足培养基需求量；中间细胞培养层较薄，且第二微孔较小，避免水凝胶材料由于芯片疏水性材质而粘附在壁上及减少水凝胶材料的用量，降低成本；基底层较薄便于后续显微镜下观察。同时该芯片设计结构集中于中间细胞培养层，避免了对准键合中可能出现的误差及结构的差异化；2. The upper liquid reservoir layer of the vascularized organoid chip provided by the present invention is thicker to meet the demand for culture medium; the middle cell culture layer is thinner, and the second micropore is smaller to avoid the hydrogel material due to the hydrophobic material of the chip. It adheres to the wall and reduces the amount of hydrogel material, reducing costs; the thin base layer facilitates subsequent observation under a microscope. At the same time, the chip design structure is concentrated on the middle cell culture layer, avoiding possible errors in alignment bonding and structural differentiation;

3、本发明提供的血管化类器官芯片为开放式设计，便于使芯片加样与自动化设备相匹配使用，同时开放式设计便于培养组织的取出进行后续的多手段分析测试；3. The vascularized organoid chip provided by the present invention has an open design, which facilitates matching the chip loading with automated equipment. At the same time, the open design facilitates the removal of cultured tissue for subsequent multi-method analysis and testing;

4、本发明提供的血管化类器官芯片可采用塑料材质，可避免传统PDMS材质对小分子药物的吸附，使后续药筛应用中结果更准确。同时该芯片可用注塑工艺量产加工，可避免传统PDMS芯片只能手工制作而无法批量生产的弊端，及手动加工中易出现的批次差；4. The vascularized organoid chip provided by the present invention can be made of plastic material, which can avoid the adsorption of small molecule drugs by traditional PDMS materials and make the results of subsequent drug screening applications more accurate. At the same time, the chip can be mass-produced by injection molding technology, which can avoid the disadvantages of traditional PDMS chips that can only be made by hand and cannot be mass-produced, as well as the batch differences that are easy to occur in manual processing;

5、本发明提供的血管化类器官芯片可采用模块化设计可适配科研工作的少量需求，以及孔板化设计满足药物筛选的高通量需求。5. The vascularized organoid chip provided by the present invention can adopt a modular design to meet the small needs of scientific research work, and a well plate design to meet the high-throughput needs of drug screening.

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

附图说明Description of drawings

本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

图1显示了本发明提供的血管化类器官芯片的结构示意图；Figure 1 shows a schematic structural diagram of the vascularized organoid chip provided by the present invention;

图2显示了本发明提供的血管化类器官芯片的剖面结构示意图；Figure 2 shows a schematic cross-sectional structural diagram of the vascularized organoid chip provided by the present invention;

图3显示了本发明提供的包含多个结构单元的血管化类器官芯片的结构示意图；Figure 3 shows a schematic structural diagram of a vascularized organoid chip including multiple structural units provided by the present invention;

图4显示了本发明提供的血管化类器官芯片进行血管模型培养的示意图；Figure 4 shows a schematic diagram of vascular model culture using the vascularized organoid chip provided by the present invention;

图5显示了流体剪切力细胞培养自动化装置的实物图；Figure 5 shows a physical diagram of the fluid shear force cell culture automation device;

图6显示了本发明实施例1中采用血管化类器官芯片生成的三维血管网络；Figure 6 shows a three-dimensional vascular network generated using a vascularized organoid chip in Embodiment 1 of the present invention;

图7显示了本发明实施例1中三维血管网络经70kD FITC-Dextran灌注的结果图；Figure 7 shows the results of perfusion of a three-dimensional vascular network with 70kD FITC-Dextran in Example 1 of the present invention;

图8显示了本发明实施例1中三维血管网络经荧光微球灌注的结果图；Figure 8 shows the results of perfusion of a three-dimensional vascular network with fluorescent microspheres in Example 1 of the present invention;

图9显示了本发明实施例2中采用血管化类器官芯片生成的血管网络包围类器官。Figure 9 shows a vascular network generated using a vascularized organoid chip in Embodiment 2 of the present invention surrounding the organoid.

附图标记：Reference signs:

100-血管化类器官芯片；100-vascularized organoid chip;

101-储液层；1011-通孔单元；10111-第一通孔；10112-第二通孔；10113-第三通孔；101-liquid storage layer; 1011-through hole unit; 10111-first through hole; 10112-second through hole; 10113-third through hole;

102-细胞培养层；1021-培养单元；10211-第一微孔；10212-第二微孔；10213-第三微孔；103-基底层。102-cell culture layer; 1021-culture unit; 10211-first micropore; 10212-second micropore; 10213-third micropore; 103-basal layer.

具体实施方式Detailed ways

下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below. The embodiments described below are illustrative and are only used to explain the present invention and are not to be construed as limitations of the present invention.

需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality" is two or more.

在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but these ranges or values are to be understood to include values approaching such ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These values The scope shall be deemed to be specifically disclosed herein.

为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。In this article, the term "comprises" or "includes" is an open-ended expression, which includes the contents specified in the present invention, but does not exclude other aspects.

在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally," "optionally," or "optionally" generally mean that the subsequently described event or condition may but need not occur, and that the description includes instances in which the event or condition occurs, and A situation in which the event or condition did not occur.

本发明第一方面提供了一种血管化类器官芯片100，如图1和图2所示，该芯片100包括依次设置的储液层101、细胞培养层102和基底层103。其中，所述储液层设置至少一个通孔单元1011，每个所述通孔单元1011包括依次排列的第一通孔10111、第二通孔10112和第三通孔10113。所述细胞培养层102设置至少一个培养单元1021，所述培养单元1021与所述通孔单元1011一一对应，每个所述培养单元1021包括依次排列的第一微孔10211、第二微孔10212和第三微孔10213，所述第一微孔10211、第二微孔10212和第三微孔10213分别与所述第一通孔10111、第二通孔10112和第三通孔10113一一对应。其中通孔单元1011中的第一通孔10111和第三通孔10113用于注入培养基，培养基将从储液层流入细胞培养层。通孔单元1011中的第二通孔10212用于注入血管化细胞和/或类器官。第二通孔10212底部形成目标高度的间隙以形成通道，所述第一微孔10211、第二微孔10212和第三微孔10213通过所述通道实现联通。A first aspect of the present invention provides a vascularized organoid chip 100. As shown in Figures 1 and 2, the chip 100 includes a liquid storage layer 101, a cell culture layer 102 and a basal layer 103 arranged in sequence. Wherein, the liquid storage layer is provided with at least one through hole unit 1011, and each of the through hole units 1011 includes a first through hole 10111, a second through hole 10112 and a third through hole 10113 arranged in sequence. The cell culture layer 102 is provided with at least one culture unit 1021. The culture unit 1021 corresponds to the through hole unit 1011. Each culture unit 1021 includes a first micropore 10211 and a second micropore arranged in sequence. 10212 and the third microhole 10213. The first microhole 10211, the second microhole 10212 and the third microhole 10213 are respectively connected with the first through hole 10111, the second through hole 10112 and the third through hole 10113. correspond. The first through hole 10111 and the third through hole 10113 in the through hole unit 1011 are used to inject culture medium, and the culture medium will flow from the liquid storage layer into the cell culture layer. The second through hole 10212 in the through hole unit 1011 is used for injecting vascularized cells and/or organoids. A gap of a target height is formed at the bottom of the second through hole 10212 to form a channel, and the first microhole 10211, the second microhole 10212 and the third microhole 10213 are connected through the channel.

其中，储液层、细胞培养层和基底层的厚度比可为(20-50):2:1。储液层厚度的设定应满足培养基需求量，可设定为5-10mm。细胞培养层厚度的设定应便于后续水凝胶材料的灌注，避免出现水凝胶粘附于孔壁而无法铺满孔底部的情况，可设定为0.2-1mm，基底层厚度的设定应便于后续通过显微镜观察，可设定为0.1-0.5mm。但需要指出的是，以上结构厚度的选择为示例性的，并不对本发明保护范围构成限制。储液层中每一个通孔单元中相邻通孔的直径应呈相异设置，且所述第二微孔的直径小于所述第二微孔对应的第二通孔的直径。优选地，第一通孔和/或第三通孔的直径大于第二通孔；更优选地，第一通孔和第三通孔的直径相同。具体地第一通孔的直径为8mm，所述第三通孔的直径为8mm，所述第二通孔的直径为5mm，所述第二微孔的直径为2mm。但需要指出的是，通孔和微孔的直径选择并不限于以上直径范围，可根据实际类器官尺寸制备需要进行合理调整。Wherein, the thickness ratio of the liquid reservoir layer, the cell culture layer and the basal layer can be (20-50):2:1. The thickness of the liquid storage layer should be set to meet the demand for the culture medium and can be set to 5-10mm. The thickness of the cell culture layer should be set to facilitate the subsequent infusion of hydrogel materials to avoid the situation where the hydrogel adheres to the well wall and cannot cover the bottom of the well. It can be set to 0.2-1mm. The thickness of the basal layer is set It should be convenient for subsequent observation through a microscope and can be set to 0.1-0.5mm. However, it should be pointed out that the above selection of structural thickness is exemplary and does not limit the scope of the present invention. The diameters of adjacent through holes in each through hole unit in the liquid storage layer should be set differently, and the diameter of the second micropore is smaller than the diameter of the second through hole corresponding to the second micropore. Preferably, the diameter of the first through hole and/or the third through hole is larger than that of the second through hole; more preferably, the diameters of the first through hole and the third through hole are the same. Specifically, the diameter of the first through hole is 8 mm, the diameter of the third through hole is 8 mm, the diameter of the second through hole is 5 mm, and the diameter of the second micropore is 2 mm. However, it should be pointed out that the diameter selection of through holes and micropores is not limited to the above diameter range, and can be reasonably adjusted according to the actual organoid size preparation needs.

根据本发明的具体的实施例，细胞培养层中第二微孔底部设定目标高度的间隙以形成通道，使一个培养单元中的三个微孔实现联通。目标高度应满足第二微孔中细胞不会溢出至两侧流道中，但不影响两侧微孔中培养基在整个流道中的流动，细胞培养层的厚度与目标高度的比可为(1.2-1.5):1。具体地，实现目标高度的结构可有多种结构形式，例如在优选情况下，如图2所示，第二微孔10212的周缘底部具有楔形流体限位结构，其宽度沿着储液层到基底层方向逐渐缩小，以减少细胞与培养基间距离，避免两侧通孔加培养基时在中间流道空气难排除而产生气泡，从而影响培养基流动性。该目标高度的选择可根据第二微孔中细胞类型进行合理选择，例如200-500μm，但并不限于此数值范围。According to a specific embodiment of the present invention, a gap of a target height is set at the bottom of the second micropore in the cell culture layer to form a channel, so that three micropores in a culture unit are connected. The target height should be such that the cells in the second microwell will not overflow into the flow channels on both sides, but will not affect the flow of the culture medium in the micropores on both sides in the entire flow channel. The ratio of the thickness of the cell culture layer to the target height can be (1.2 -1.5):1. Specifically, the structure to achieve the target height can have a variety of structural forms. For example, in a preferred case, as shown in Figure 2, the peripheral bottom of the second micropore 10212 has a wedge-shaped fluid limiting structure, the width of which is along the liquid storage layer to The direction of the basal layer gradually shrinks to reduce the distance between the cells and the culture medium, so as to avoid air bubbles being generated in the middle flow channel when the medium is added to the through holes on both sides, thus affecting the fluidity of the culture medium. The target height can be selected reasonably according to the type of cells in the second microwell, for example, 200-500 μm, but is not limited to this numerical range.

根据本发明的具体的实施例，储液层、细胞培养层和基底层可键合得到血管化类器官芯片，储液层的材质可选择PMMA、PS、COC，细胞培养层的材质可选择PMMA、PS、PC，基底层的材质可选择玻璃、PC、PS以满足观察细胞培养层10212内类器官或血管的生成情况的需求。According to specific embodiments of the present invention, the liquid storage layer, the cell culture layer and the basal layer can be bonded to obtain a vascularized organoid chip. The material of the liquid storage layer can be selected from PMMA, PS, and COC, and the material of the cell culture layer can be selected from PMMA. , PS, PC, the material of the basal layer can be selected from glass, PC, and PS to meet the needs of observing the formation of organoids or blood vessels in the cell culture layer 10212.

根据本发明的具体的实施例，血管化类器官芯片可设计为包含多个结构单元的单元模块化，如图3所示，其包含32个通孔单元，每个通孔单元可分别培养各自的类器官，满足不同的实验需求。According to specific embodiments of the present invention, the vascularized organoid chip can be designed as a modular unit containing multiple structural units. As shown in Figure 3, it contains 32 through-hole units, and each through-hole unit can be cultured separately. Organoids to meet different experimental needs.

本发明还提供了一种血管模型的制备方法，该制备方法基于前述的血管化类器官芯片，包括将血管化细胞与水凝胶材料混合后注入第二通孔，分别向所述第一通孔与第三通孔中的至少一个和所述第二通孔注入培养基，进行培养。其中血管化细胞可选择人脐静脉内皮细胞、人肺成纤维细胞等，水凝胶材料可选择明胶或基质胶等。具体地，可选择人脐静脉内皮细胞(HUVEC)及人肺成纤维细胞(NHLF)以一定比例在纤维蛋白原、凝血酶、基质胶中混合均匀后注入第二通孔，待基质胶凝固后从两侧通孔中的一侧注入培养基，并向第二通孔注入一定量培养基，由于液面高度差，培养基将通过通道流至另一侧通孔中，直至两侧通孔中液体高度一致，如图4所示。由于表面张力，第二通孔中的细胞外基质将聚集在中间通孔而不会溢出至两侧流道中。The present invention also provides a method for preparing a blood vessel model. The preparation method is based on the aforementioned vascularized organoid chip and includes mixing vascularized cells and hydrogel material and then injecting them into the second through hole, respectively injecting them into the first through hole. At least one of the hole and the third through hole and the second through hole are injected with culture medium for culture. Among them, the vascularized cells can be human umbilical vein endothelial cells, human lung fibroblasts, etc., and the hydrogel materials can be gelatin or Matrigel. Specifically, human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (NHLF) can be mixed with fibrinogen, thrombin, and Matrigel in a certain proportion and then injected into the second through hole. After the Matrigel has solidified, Inject culture medium from one side of the through holes on both sides, and inject a certain amount of culture medium into the second through hole. Due to the difference in liquid level, the culture medium will flow through the channel into the through holes on the other side until it reaches the through holes on both sides. The liquid height in the medium is consistent, as shown in Figure 4. Due to surface tension, the extracellular matrix in the second through hole will gather in the middle through hole and will not overflow into the flow channels on both sides.

本发明还提供了一种血管化类器官的培养方法，该方法基于前述的血管化类器官芯片，包括将血管化细胞、类器官与水凝胶材料混合后注入所述第二通孔，分别向所述第一通孔与第三通孔中的至少一个和所述第二通孔注入培养基，进行培养。其中血管化细胞可选择人脐静脉内皮细胞、人肺成纤维细胞等，类器官包括选自肿瘤类器官、胰腺类器官、肝脏类器官、肾脏类器官、肠类器官、胃类器官中的至少一种，肿瘤类器官包括选自肠癌类器官、肺癌类器官、胃癌类器官、胰腺癌类器官中的至少一种，水凝胶材料可选择明胶或基质胶等。具体地，可将类器官、人脐静脉内皮细胞及人肺成纤维细胞以一定比例在纤维蛋白原、凝血酶及基质胶中混合均匀后注入第二通孔，第二通孔中的细胞外基质凝固后，向三个通孔中注入培养基进行培养。培养一段时间后，还可将该芯片放置于流体剪切力细胞培养自动化装置或半自动化装置上，如图5所示，该设备倾斜产生液面高度差，培养基将通过流道流至另一侧通孔中，直至两侧通孔中液体高度一致，再反向倾斜，继续产生流体剪切力刺激。在培养过程中利用两侧通孔中的液面高度差产生的流体剪切力刺激，HUVEC细胞自发生长形成三维血管网络。血管网络在动态条件下培养10天后形成可灌注血管网络。The present invention also provides a method for cultivating vascularized organoids. The method is based on the aforementioned vascularized organoid chip and includes mixing vascularized cells, organoids and hydrogel materials and then injecting them into the second through hole, respectively. Inject culture medium into at least one of the first through hole, the third through hole and the second through hole for culturing. Among them, the vascularized cells can be human umbilical vein endothelial cells, human lung fibroblasts, etc., and the organoids include at least one selected from the group consisting of tumor organoids, pancreatic organoids, liver organoids, kidney organoids, intestinal organoids, and gastric organoids. In one embodiment, the tumor organoids include at least one selected from the group consisting of intestinal cancer organoids, lung cancer organoids, gastric cancer organoids, and pancreatic cancer organoids, and the hydrogel material can be gelatin or Matrigel. Specifically, organoids, human umbilical vein endothelial cells, and human lung fibroblasts can be mixed evenly with fibrinogen, thrombin, and Matrigel in a certain proportion and then injected into the second through hole. After the matrix is solidified, medium is injected into the three through holes for culture. After culturing for a period of time, the chip can also be placed on a fluid shear cell culture automated device or semi-automatic device, as shown in Figure 5. The tilt of the device creates a height difference in the liquid level, and the culture medium will flow through the flow channel to another area. In the through hole on one side, until the height of the liquid in the through holes on both sides is the same, it is then tilted in the opposite direction to continue to generate fluid shear force stimulation. During the culture process, the fluid shear force generated by the liquid level difference in the through holes on both sides is stimulated, and HUVEC cells grow spontaneously to form a three-dimensional vascular network. The vascular network formed a perfusable vascular network after being cultured under dynamic conditions for 10 days.

下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

实施例1血管化类器官芯片用于生成三维血管网络Example 1 Vascularized organoid chip used to generate three-dimensional vascular network

准备人脐静脉内皮细胞与人肺成纤维细胞培养于T25细胞培养瓶，待细胞汇合度达到100％时，胰酶消化，计数。分别取8×106个人脐静脉内皮细胞与4×106个人肺成纤维细胞混合于15ml离心管，转速300×g离心5min。小心吸弃上清，加入提前准备好的浓度为10mg/ml纤维蛋白原凝胶，混合均匀，置于冰上待用。用内皮细胞完全培养基将10U/ml的凝血酶稀释为1U/ml，以10μL每份分装于1.5ml EP管，置于冰上待用。取10μL含有细胞的纤维蛋白原凝胶与其中一份含有凝血酶的完全培养基混合均匀，灌注到芯片的第二通孔，于37℃细胞培养箱凝固20min。凝固后在两侧通孔加入内皮细胞完全培养基，将芯片放置于配套设备上，设备倾斜产生液面高度差，培养基将通过流道流至另一侧通孔中，直至两侧通孔中液体高度一致，再反向倾斜，继续产生流体刺激。培养10天后产生可灌注的三维血管网络，如图6所示。70kD FITC-Dextran灌注显示，血管具有良好的可灌注性，如图7所示。此外，使用荧光微球进行灌注，如图8所示，荧光微球可以顺着血管内壁从血管网络一侧到达另一侧，说明血管已形成闭合管状网络结构，具有良好的可灌注性。Prepare human umbilical vein endothelial cells and human lung fibroblasts to be cultured in T25 cell culture flasks. When the confluence of the cells reaches 100%, they are digested with trypsin and counted. Mix 8×10 6 human umbilical vein endothelial cells and 4×10 6 human lung fibroblasts in a 15ml centrifuge tube, and centrifuge at 300×g for 5 minutes. Carefully aspirate and discard the supernatant, add the 10 mg/ml fibrinogen gel prepared in advance, mix evenly, and place on ice for later use. Dilute 10 U/ml thrombin to 1 U/ml with complete endothelial cell culture medium, aliquot 10 μL each into a 1.5 ml EP tube, and keep on ice until use. Take 10 μL of fibrinogen gel containing cells and mix it evenly with one part of the complete culture medium containing thrombin, perfuse it into the second through hole of the chip, and solidify in a 37°C cell culture incubator for 20 minutes. After solidification, add complete endothelial cell culture medium to the through holes on both sides, place the chip on the supporting equipment, tilt the equipment to create a liquid level difference, and the culture medium will flow through the flow channel to the through hole on the other side, until it reaches the through holes on both sides. The height of the liquid in the middle is the same, and then tilted in the opposite direction to continue to produce fluid stimulation. A perfusable three-dimensional vascular network was produced after 10 days of culture, as shown in Figure 6 . 70kD FITC-Dextran perfusion showed that the blood vessels had good perfusability, as shown in Figure 7. In addition, fluorescent microspheres were used for perfusion. As shown in Figure 8, the fluorescent microspheres could travel along the inner wall of the blood vessels from one side of the vascular network to the other, indicating that the blood vessels had formed a closed tubular network structure and had good perfusionability.

实施例2血管化类器官芯片用于血管类器官共培养Example 2 Vascularized organoid chip for co-culture of vascularized organoids

准备1000个左右100μm左右大小的肠癌类器管，转移到15ml离心管，加入5ml的类器官回收液在冰上轻轻晃动30min将基质胶去除，加入DPBS定容至10ml，300×g离心5min，弃上清，加入100μL基质胶吹打混匀置于冰上待用。将血管内皮完全培养基与肠癌类器官培养基按照1：1的比例混合，待用。将10mg/ml纤维蛋白原与含有类器官的基质胶按照9:1的比例混合，置于冰上待用。准备10U/ml的凝血酶，将其以2μL每份分装于1.5ml EP管置于冰上待用。准备8×106个人脐静脉内皮细胞与4×106个人肺成纤维细胞混合于15ml离心管，转速300×g离心5min。小心吸弃上清，加入提前准备好的含有类器官的混合胶，混合均匀，置于冰上待用。取8μL混合胶与分装好的凝血酶吹打2-3次混合，迅速灌注于芯片的第二通孔，37℃培养箱放置10min使其凝固。凝固后在两侧通孔加入混合后的培养基，将芯片放置于配套设备上，设备倾斜产生液面高度差，培养基将通过流道流至另一侧通孔中，直至两侧通孔中液体高度一致，再反向倾斜，继续产生流体剪切力刺激。3天可见内皮细胞逐渐形成血管网络包围类器官(图9)。Prepare about 1,000 intestinal cancer organoid tubes with a size of about 100 μm, transfer them to a 15 ml centrifuge tube, add 5 ml of organoid recovery solution, shake gently on ice for 30 minutes to remove the Matrigel, add DPBS to adjust the volume to 10 ml, and centrifuge at 300×g After 5 minutes, discard the supernatant, add 100 μL of Matrigel, mix by pipetting, and place on ice until use. Mix vascular endothelial complete culture medium and intestinal cancer organoid culture medium at a ratio of 1:1 and set aside. Mix 10 mg/ml fibrinogen and organoid-containing Matrigel at a ratio of 9:1 and place on ice until use. Prepare 10 U/ml thrombin, aliquot 2 μL into 1.5 ml EP tubes and keep on ice until use. Prepare a mixture of 8 × 10 6 human umbilical vein endothelial cells and 4 × 10 6 human lung fibroblasts in a 15 ml centrifuge tube, and centrifuge at 300 × g for 5 minutes. Carefully discard the supernatant, add the mixed gel containing organoids prepared in advance, mix evenly, and place on ice until use. Take 8 μL of the mixed gel and the aliquoted thrombin by pipetting 2-3 times to mix, quickly pour it into the second through hole of the chip, and place it in a 37°C incubator for 10 minutes to solidify. After solidification, add the mixed culture medium to the through holes on both sides, place the chip on the supporting equipment, and tilt the equipment to create a liquid level difference. The culture medium will flow through the flow channel to the through holes on the other side, until it reaches the through holes on both sides. The height of the liquid in the middle is the same, and then it is tilted in the opposite direction to continue to generate fluid shear force stimulation. After 3 days, it was seen that endothelial cells gradually formed a vascular network surrounding the organoids (Figure 9).

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (15)

1. The vascularized organoid chip is characterized by comprising a liquid storage layer, a cell culture layer and a basal layer which are sequentially arranged;
the liquid storage layer is provided with at least one through hole unit, each through hole unit comprises a first through hole, a second through hole and a third through hole which are sequentially arranged, and the diameters of the adjacent through holes are arranged differently;
the cell culture layer is provided with at least one culture unit, the culture units are in one-to-one correspondence with the through hole units, each culture unit comprises a first micropore, a second micropore and a third micropore which are sequentially arranged, the first micropore, the second micropore and the third micropore are respectively in one-to-one correspondence with the first through hole, the second through hole and the third through hole, gaps with target heights are formed at the bottom of the second micropore so as to form a channel, and the first micropore, the second micropore and the third micropore are communicated through the channel.
2. The vascularized organoid chip of claim 1, wherein the thickness ratio of the reservoir layer, cell culture layer, and basal layer is (20-50): 2:1;
optionally, the thickness of the liquid storage layer is 5-10mm;
optionally, the cell culture layer has a thickness of 0.2-1mm;
optionally, the base layer has a thickness of 0.1-0.5mm;
optionally, the ratio of the thickness of the cell culture layer to the target height is (1.2-1.5): 1;
optionally, the target height is 200-500 μm;
optionally, the peripheral bottom of the second microwell has a wedge-shaped structure to form a gap of a target height, the width of the wedge-shaped structure gradually shrinking along the direction from the liquid storage layer to the basal layer.
3. The vascularized organoid chip of claim 1, wherein the first and/or third through holes have a larger diameter than the second through hole;
optionally, the diameters of the first through hole and the third through hole are the same;
optionally, the diameter of the second microwells is less than the diameter of the second through holes;
optionally, the diameter ratio of the first through hole, the second through hole and the third through hole is 8:5:8;
optionally, the diameter of the first through hole is 8mm, the diameter of the second through hole is 5mm, and the diameter of the third through hole is 8mm;
optionally, the diameter of the second microwells is 2mm.
4. The vascularized organoid chip of claim 1, wherein the cell culture layer is in contact with the base layer.
5. The vascularized organoid chip of claim 1, wherein the material of the reservoir layer is one or more of PMMA, PS, COC;
optionally, the material of the cell culture layer is one or more of PMMA, PS, PC;
optionally, the substrate layer is made of one or more of glass, PC and PS.
6. Use of the vascularized organoid chip of any of claims 1-5 for the preparation of a vascular model or vascularized organoid.
7. A method for preparing a vascular model, wherein the method for preparing the vascularized organoid chip according to any one of claims 1 to 5 comprises mixing vascularized cells with a hydrogel material, injecting the mixture into the second through hole, and injecting a culture medium into at least one of the first through hole and the third through hole and the second through hole, respectively, for culturing.
8. The method of claim 7, wherein the vascularized cells comprise human umbilical vein endothelial cells, human lung fibroblasts;
optionally, the hydrogel material comprises a material selected from gelatin or matrigel.
9. The method of claim 7, further comprising placing the vascularized organoid chip in a fluid shear cell culture automation device or a semi-automation device.
10. A method for preparing a vascularized organoid, wherein the method for preparing a vascularized organoid chip according to any one of claims 1 to 5 comprises mixing vascularized cells, organoids and hydrogel material and injecting the mixture into the second through-hole, and injecting a culture medium into at least one of the first through-hole and the third through-hole and the second through-hole, respectively, for culturing.
11. The method of claim 10, wherein the vascularized cells comprise human umbilical vein endothelial cells, human lung fibroblasts;
optionally, the organoids comprise at least one selected from the group consisting of tumor organoids, pancreatic organoids, liver organoids, kidney organoids, intestinal organoids, and stomach organoids;
optionally, the tumor organoid comprises at least one selected from the group consisting of intestinal cancer organoids, lung cancer organoids, gastric cancer organoids, pancreatic cancer organoids;
optionally, the organoids comprise a member selected from the group consisting of intestinal cancer organoids;
optionally, the hydrogel material comprises a material selected from gelatin or matrigel.
12. The method of claim 10, further comprising placing the vascularized organoid chip in a fluid shear cell culture automation device or a semi-automation device.
13. A vascular model, characterized in that it is obtained by the preparation method according to any one of claims 7-9.
14. A vascularized organoid, characterized in that it is obtained by the preparation method according to any one of claims 10-12.
15. Use of a vascular model as claimed in claim 13 or a vascularised organoid as claimed in claim 14 in drug screening.
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