WO2022001631A1

WO2022001631A1 - Device for preparing cell clusters, construction method therefor and application thereof

Info

Publication number: WO2022001631A1
Application number: PCT/CN2021/099718
Authority: WO
Inventors: 姚睿; 苏奕君; 梁少君; 薛胜楠; 申函宁
Original assignee: 清华大学
Priority date: 2020-06-29
Filing date: 2021-06-11
Publication date: 2022-01-06
Also published as: CN113930337B; CN113930337A

Abstract

The present application provides a device for preparing cell clusters, a construction method therefor and an application thereof. The device comprises a cell culture plate, and a substrate that is fixed in a well of the cell culture plate and has a microcavity array. Each microcavity forms a space for cell cluster growth, and may also have a non-vertical wall surface. Cell cluster properties are obtained by means of shape and volume regulation. The preparation method therefor comprises: preparing a size-adjustable microcavity template, and then obtaining a substrate having a microcavity array by means of two-step rollover. The device provided by the present application can be used for preparing cell clusters in a large scale conveniently, and can be used repeatedly. The elastic modulus of the substrate can be adjusted to meet the requirements of co-culture of various types of cells and multiple types of cells. The cell clusters prepared using this device have high activity, uniform size, complete shape, controllable performance, and good biological properties, and can be used in cell therapy, high-throughput model construction, multi-cell co-culture, stem cell differentiation, organoid construction, drug screening, tissue regeneration, and in vitro artificial systems.

Description

用于制备细胞团簇的装置及其构建方法及应用Device for preparing cell clusters, construction method and application thereof

相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本申请要求于2020年6月29日提交的申请号为202010610263.X，发明名称为“用于制备细胞团簇的装置及其构建方法及应用”的中国专利申请的优先权，其通过引用方式全部并入本文。This application claims the priority of the Chinese patent application filed on June 29, 2020 with the application number 202010610263.X and the invention titled "Device for Preparing Cell Clusters and Its Construction Method and Application", which is by way of reference All incorporated herein.

技术领域technical field

本申请涉及生物技术和组织工程技术领域，具体地说，涉及一种用于制备细胞团簇的装置及其构建方法及应用。The present application relates to the technical fields of biotechnology and tissue engineering, and in particular, to a device for preparing cell clusters, a construction method and applications thereof.

背景技术Background technique

传统的细胞培养方式是平面培养，采用平面培养得到的单层细胞培养与体内细胞的生理结构并不相同，会影响细胞的存活与功能。利用细胞间的自组装形成细胞团簇，可以构建一个准3D环境。与平面培养相比，三维细胞培养可以重建细胞与细胞、细胞与基质之间的相互作用，减少体外培养与天然组织之间的差异。例如，胚胎干细胞、间充质干细胞、多种肿瘤细胞等细胞具有成团的特性。此外，胰岛β细胞需要形成细胞团簇以维持其功能，构建细胞团簇有利于细胞的培养和分化。同时，有关类器官的研究近来也快速增加，而类器官往往需要多种细胞成团共培养，以保证其结构发育成形和功能表达。细胞团簇的下游应用有药物检测、体内移植等，这些下游应用一般都需要10 ⁸～10 ⁹个细胞才能得到可靠的结果，因此前期制备大量的细胞团簇。鉴于细胞团簇的广泛应用，大规模制备细胞团簇是非常有必要的。 The traditional cell culture method is flat culture. The monolayer cell culture obtained by flat culture is not the same as the physiological structure of cells in vivo, which will affect the survival and function of cells. Using the self-assembly of cells to form cell clusters, a quasi-3D environment can be constructed. Compared with flat culture, 3D cell culture can recreate cell-cell, cell-matrix interactions and reduce the differences between in vitro culture and native tissues. For example, cells such as embryonic stem cells, mesenchymal stem cells, and various tumor cells have the characteristic of clumps. In addition, pancreatic β cells need to form cell clusters to maintain their functions, and the construction of cell clusters is beneficial to cell culture and differentiation. At the same time, research on organoids has also increased rapidly recently, and organoids often require co-culture of multiple cells to ensure their structural development and functional expression. The downstream applications of cell clusters include drug detection, in vivo transplantation, etc. These downstream applications generally require 10 ⁸ to 10 ⁹ cells to obtain reliable results, so a large number of cell clusters are prepared in the early stage. In view of the wide application of cell clusters, large-scale preparation of cell clusters is very necessary.

构建细胞团簇的一个重要因素是团簇尺寸，过小不利于细胞成团，过大不利于细胞团内部的营养供给，进而造成细胞团簇内部的异质性，因此制备尺寸可控的细胞团簇至关重要。另一个重要因素是基底的刚度，不同细胞种类对于材料刚度的要求不同，因此在制备细胞团簇时需要有一个合适的培养基底。An important factor in the construction of cell clusters is the size of the clusters. Too small is not conducive to the formation of cells, and too large is not conducive to the nutrient supply inside the cell clusters, thereby causing heterogeneity within the cell clusters. Therefore, cells with controllable size are prepared. Clusters are critical. Another important factor is the stiffness of the substrate. Different cell types have different requirements for material stiffness, so a suitable culture substrate is required when preparing cell clusters.

目前有关细胞团簇的研究，一般是在常规多孔培养皿中铺上基底，种植一定数量的细胞后依靠细胞自组装形成团簇。这种方法虽然可以提供有利于细胞成团和培养的软底，但数量、尺寸受限，难以获得大量、均一的细胞团簇。引入具有可控尺寸的微腔阵列，可以解决这一问题，得到尺寸均一、数量可观的细胞团簇。At present, the research on cell clusters generally involves laying a substrate in a conventional multi-well culture dish and planting a certain number of cells to form clusters by self-assembly of cells. Although this method can provide a soft bottom that is beneficial for cell aggregation and culture, it is limited in quantity and size, and it is difficult to obtain a large number of uniform cell clusters. The introduction of microcavity arrays with controllable dimensions can solve this problem and obtain cell clusters of uniform size and considerable number.

目前有部分商业化产品，多孔培养皿底部具备微腔阵列，可以制备大量均一的细胞团簇，但是塑料基底，刚度太大，微孔尺寸和形状不能满足自定义的要求，且无法重复使用，成本和费用过高。At present, there are some commercial products. The bottom of the multi-well culture dish is equipped with a microcavity array, which can prepare a large number of uniform cell clusters. However, the plastic substrate is too rigid, and the size and shape of the micropores cannot meet the custom requirements, and cannot be reused. Costs and fees are exorbitant.

此外也有部分研究和专利制备微腔阵列用以培养细胞团簇。常见的方法是通过模板法制备聚二甲基硅氧烷***，再利用***制备出微腔阵列，用以培养细胞。这种方法制备得到的多是PEG、琼脂等水凝胶基底，力学性能调节范围有限，且无法重复使用。In addition, there are also some studies and patents to prepare microcavity arrays for culturing cell clusters. A common method is to prepare polydimethylsiloxane stamps by a template method, and then use the stamps to prepare microcavity arrays for culturing cells. Most of the hydrogel substrates prepared by this method are hydrogel substrates such as PEG and agar, which have a limited range of mechanical properties and cannot be reused.

还有研究采用PDMS作为细胞培养的基底材料，方法是通过光刻技术得到垂直壁面的微图案模板，再用模板法制备出微腔。这种方法仅能制备出垂直壁面的微腔，难以得到非垂直壁面，在细胞聚集体的产生大程度依靠细胞间作用力。There are also studies using PDMS as the substrate material for cell culture. The method is to obtain a micro-pattern template with a vertical wall by photolithography, and then prepare a micro-cavity by a template method. This method can only prepare microcavities with vertical walls, and it is difficult to obtain non-vertical walls. The generation of cell aggregates largely depends on the intercellular force.

综上，大规模制备细胞团簇仍然存在着挑战，目前亟需可同时满足(1)可调力学性能、(2)非垂直壁面、(3)可重复使用，这三个条件的细胞培养环境。制备可调力学性能的基底，通过调节交联过程，满足不同细胞对基质刚度的需求；非垂直壁面除了具有和垂直壁面相同的空间限制的作用外，还可以利用细胞自身的重力促进细胞聚集成团；重复使用可以节省制备的成本和时间。开发同时满足适合刚度的基底、自定义形状微腔(垂直壁面或非垂直壁面)、可重复使用三个要求的培养装置和技术，从而有效产生大规模活性高的细胞团簇，成为当务之急。To sum up, there are still challenges in the large-scale preparation of cell clusters, and a cell culture environment that can simultaneously satisfy the three conditions of (1) adjustable mechanical properties, (2) non-vertical walls, and (3) reusability is urgently needed. . Fabrication of substrates with adjustable mechanical properties, and by adjusting the cross-linking process, to meet the needs of different cells for matrix stiffness; in addition to having the same spatial confinement role as vertical walls, non-vertical walls can also utilize the cell's own gravity to promote cell aggregation. group; reuse can save cost and time of preparation. It is imperative to develop culture devices and techniques that simultaneously satisfy the three requirements of substrates with suitable stiffness, custom-shaped microcavities (vertical or non-vertical walls), and reusability, so as to efficiently generate large-scale and highly active cell clusters.

发明内容SUMMARY OF THE INVENTION

本申请的目的是提供一种用于制备细胞团簇的装置及其构建方法及应用。The purpose of the present application is to provide a device for preparing cell clusters and a construction method and application thereof.

为了实现本申请目的，第一方面，本申请提供一种用于制备细胞团簇的装置，所述装置包括细胞培养板以及固定于细胞培养板的微孔内且具有微腔阵列的基底。In order to achieve the purpose of the present application, in a first aspect, the present application provides a device for preparing cell clusters, the device comprising a cell culture plate and a substrate fixed in a microwell of the cell culture plate and having a microcavity array.

每个微腔构成细胞团簇生长的空间(大致决定细胞团簇生长的尺寸和空间)；微腔的形状可选自圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形等任意规则或不规则形状，尤其以倒锥形这类非垂直壁面为优先项；微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，基底上排列的微腔的数量为1000～5000个左右(视微腔尺寸与模板整体尺寸而定)，阵列图案和尺寸可相同，也可不同，排列可整齐均一，也可自定义排列，同一基底阵列可包含不同形状、尺寸和排列方式的微腔。 Each microcavity constitutes a space for cell cluster growth (roughly determines the size and space of cell cluster growth); the shape of the microcavity can be selected from cylindrical, square, rectangular, triangular, diamond, Any regular or irregular shape such as hexagonal prism, inverted cone, etc., especially the non-vertical wall surface such as inverted cone is the priority; the upper surface area of the microcavity is 0.04～1mm ² , the depth of the microcavity is 100～500μm, and the substrate The number of microcavities arranged on the top is about 1000 to 5000 (depending on the size of the microcavity and the overall size of the template). Arrays can contain microcavities of different shapes, sizes, and arrangements.

本申请提供的装置示意图见图1。微腔示意图见图2。The schematic diagram of the device provided in this application is shown in FIG. 1 . The schematic diagram of the microcavity is shown in Figure 2.

用于制作基底的材料可选自聚丙烯、聚苯乙烯、聚丙烯酰胺、聚乳酸、聚羟基酸、聚乳酸醇酸共聚物、聚二甲基硅氧烷(PDMS)、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、聚氧化乙烯、丝素蛋白、丝素蛋白衍生物、壳聚糖、明胶、明胶衍生物、藻酸盐、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白和纤维蛋白等中的至少一种，优选聚二甲基硅氧烷和丝素蛋白的混合物。更优选地，聚二甲基硅氧烷和丝素蛋白的质量比为10:1～1:10。The material used to make the substrate can be selected from polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane (PDMS), polyanhydride, polyacid Ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, silk fibroin , silk fibroin derivatives, chitosan, gelatin, gelatin derivatives, alginate, agar, matrigel, collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, cellulose, cellulose-derived materials, At least one of proteoglycan, proteoglycan derivatives, glycoproteins, glycoprotein derived materials, laminin, fibronectin and fibrin, etc., preferably a mixture of polydimethylsiloxane and silk fibroin. More preferably, the mass ratio of polydimethylsiloxane and silk fibroin is 10:1-1:10.

所述细胞培养板为商用多孔培养板，优选6孔板、12孔板或24孔板。The cell culture plate is a commercial multi-well culture plate, preferably a 6-well plate, a 12-well plate or a 24-well plate.

在一个实施例中，所述基底的弹性模量为0.1MPa～10MPa，可根据微腔基底材料的成分、浓度和固化特性调节弹性模量。In one embodiment, the elastic modulus of the substrate is 0.1 MPa to 10 MPa, and the elastic modulus can be adjusted according to the composition, concentration and curing characteristics of the microcavity substrate material.

在一个实施例中，所述基底的pH值为3～12。In one embodiment, the pH of the substrate is 3-12.

在一个实施例中，所述基底的厚度为100μm～2cm。In one embodiment, the thickness of the substrate is 100 μm˜2 cm.

进一步地，为保证细胞团簇的稳定粘附，可根据需要添加有利于细胞团簇粘附和生长的细胞外基质成分，如胶原、基质胶、蛋白多糖、糖蛋白、透明质酸、层连接蛋白或纤维连接蛋白等。Further, in order to ensure the stable adhesion of cell clusters, extracellular matrix components that are conducive to the adhesion and growth of cell clusters can be added as needed, such as collagen, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminar connection protein or fibronectin, etc.

在一个实施例中，所述细胞外基质材料的质量百分比浓度为0.1％～80％，优选1％～25％。In one embodiment, the mass percentage concentration of the extracellular matrix material is 0.1%-80%, preferably 1%-25%.

本装置可重复使用，一次使用、收集细胞团簇后可进行清洗、灭菌并再次使用，且能保持微图形精度和无菌程度。The device is reusable, and can be cleaned, sterilized and used again after one use, cell clusters are collected, and the micro-pattern accuracy and sterility can be maintained.

第二方面，本申请提供一种用于制备细胞团簇的装置的构建方法，包括：先制备尺寸可调、形状可自定义的微图形模板，再通过两步模板法得到具有微腔阵列的基底。具体如下：In a second aspect, the present application provides a method for constructing a device for preparing cell clusters, including: firstly preparing a micro-pattern template with adjustable size and customizable shape, and then obtaining a micro-pattern template with a micro-cavity array by a two-step template method. base. details as follows:

1)采用干法蚀刻、湿法蚀刻或光刻工艺制作具有微腔阵列的模板；1) Use dry etching, wet etching or photolithography to make a template with a microcavity array;

2)制备翻模***。对于液态或半固态的***材料，利用模板法，将***材料覆盖在模板上，固化成型后从模板上剥离，得到翻模***。对于热塑性固态材料，利用热压法，将模具微图案与***材料(如聚甲基丙烯酸甲酯)贴合后加压、加热，再机械或化学剥离后，得到翻模***。2) Preparation of stamps. For the liquid or semi-solid stamp material, the stamp material is covered on the template by using the template method, and then peeled off from the template after curing and forming, to obtain the over-molded stamp. For thermoplastic solid materials, using the hot pressing method, the mold micro-pattern and the stamp material (such as polymethyl methacrylate) are laminated, pressurized, heated, and then mechanically or chemically peeled off to obtain a stamped stamp.

3)将基底材料与交联剂混合(要求进行抽真空操作去除气泡)，浇铸在翻模***表面，进行消泡处理后固化成型；3) Mix the base material with the cross-linking agent (requires vacuuming to remove air bubbles), cast it on the surface of the stamp, and cure and form after defoaming;

4)将翻模***从上述成型材料中机械剥离，或者通过化学或物理方法从上述成型材料中去除翻模***，得到具有微腔阵列的基底；4) mechanically peeling off the mold-reversing seal from the above-mentioned molding material, or removing the mold-reversing seal from the above-mentioned molding material by chemical or physical methods to obtain a substrate with a microcavity array;

5)将基底固定于细胞培养板的微孔内，灭菌处理(高温灭菌或紫外灭菌)。5) Fix the substrate in the microwell of the cell culture plate, and sterilize it (high temperature sterilization or ultraviolet sterilization).

步骤3)中所述的基底材料可选自聚丙烯、聚苯乙烯、聚丙烯酰胺、聚乳酸、聚羟基酸、聚乳酸醇酸共聚物、聚二甲基硅氧烷(PDMS)、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、聚氧化乙烯、丝素蛋白、丝素蛋白衍生物、壳聚糖、明胶、明胶衍生物、藻酸盐、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白和纤维蛋白等中的至少一种，优选聚二甲基硅氧烷和丝素蛋白的混合物。更优选地，聚二甲基硅氧烷和丝素蛋白的质量比为10:1～1:10。The base material described in step 3) can be selected from polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane (PDMS), polyanhydride , Polyester, Polyamide, Polyamino acid, Polyacetal, Polycyanoacrylate, Polyurethane, Polypyrrole, Polyester, Polymethacrylate, Polyethylene, Polycarbonate, Polyethylene oxide, Silk Fibroin, Silk Fibroin Derivatives, Chitosan, Gelatin, Gelatin Derivatives, Alginate, Agar, Matrigel, Collagen, Collagen Derivatives, Hyaluronic Acid, Hyaluronic Acid Derivatives, Cellulose, Cellulose At least one of derived materials, proteoglycans, proteoglycan derivatives, glycoproteins, glycoprotein derived materials, laminin, fibronectin and fibrin, etc., preferably a mixture of polydimethylsiloxane and silk fibroin . More preferably, the mass ratio of polydimethylsiloxane and silk fibroin is 10:1-1:10.

步骤1)中所述模板上的微腔形状可选自圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形等任意规则或不规则形状，尤其以倒锥形这类非垂直壁面为优先项；微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，模板上排列的微腔的数量为1000～5000个。 The shape of the microcavity on the template described in step 1) can be selected from any regular or irregular shape such as cylinder, square column, rectangular column, triangular column, diamond column, hexagonal column, inverted cone, etc., especially The non-vertical wall surface such as inverted cone is the priority; the upper surface area of the microcavity is 0.04-1 mm ² , the depth of the micro-cavity is 100-500 μm, and the number of micro-cavities arranged on the template is 1000-5000.

步骤1)中用于制作模板的材料可选自硅、铝、铁、锡、玻璃、聚甲基丙烯酸甲酯、聚二甲基硅氧烷、聚己内酯、聚三亚甲基碳酸酯、聚四氟乙烯、聚氧化乙烯、聚乙烯醋酸乙烯酯、聚对二氧环己酮、聚醚醚酮等中的至少一种。The material used for making the template in step 1) can be selected from silicon, aluminum, iron, tin, glass, polymethyl methacrylate, polydimethylsiloxane, polycaprolactone, polytrimethylene carbonate, At least one of polytetrafluoroethylene, polyethylene oxide, polyethylene vinyl acetate, polydioxanone, polyether ether ketone, and the like.

步骤2)中所述的***材料为可降解材料或不可降解材料，所述可降解材料可选自明胶、明胶衍生物、琼脂、琼脂糖、

F-127、聚乙烯醇、聚乙二醇等中的至少一种，所述不可降解材料可选自聚甲基丙烯酸甲酯、环氧树脂、酚醛树脂、聚氯乙烯树脂、不饱和聚酯树脂、石膏、硅胶(硅胶基质)等中的至少一种。 The seal material described in step 2) is a degradable material or a non-degradable material, and the degradable material can be selected from gelatin, gelatin derivatives, agar, agarose,

At least one of F-127, polyvinyl alcohol, polyethylene glycol, etc., the non-degradable material can be selected from polymethyl methacrylate, epoxy resin, phenolic resin, polyvinyl chloride resin, unsaturated polyester At least one of resin, gypsum, silica gel (silica gel matrix), and the like.

在一个实施例中，步骤2)中所述***材料的质量百分比浓度为0.1％～80％，优选1％～25％。In one embodiment, the mass percentage concentration of the seal material in step 2) is 0.1%-80%, preferably 1%-25%.

在一个实施例中，步骤3)中所述交联剂的浓度为0.1mM～10M，优选1mM～100mM。In one embodiment, the concentration of the cross-linking agent in step 3) is 0.1 mM to 10 M, preferably 1 mM to 100 mM.

所述交联剂可选自含氢硅油、硅烷偶联剂、二价阳离子、京尼平、戊二醛、已二酸二酰肼、环氧氯丙烷、碳化二亚胺、凝血酶及其衍生物等中的至少一种，优选含氢硅油。The cross-linking agent can be selected from hydrogen-containing silicone oil, silane coupling agent, divalent cation, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide, thrombin and the like. At least one of derivatives, etc., preferably hydrogen-containing silicone oil.

在一个实施例中，步骤3)中所述基底材料与交联剂按1000:1～1:1000的体积比混合，优选10:1～1:10。In one embodiment, the base material and the cross-linking agent in step 3) are mixed in a volume ratio of 1000:1 to 1:1000, preferably 10:1 to 1:10.

在一个实施例中，步骤3)中固化的条件为：温度10～100℃和/或光照强度0.5～1000lx。In one embodiment, the conditions for curing in step 3) are: a temperature of 10-100° C. and/or a light intensity of 0.5-1000 lx.

在本申请的一个具体实施方式中，用于制备细胞团簇的装置的构建方法包括：In a specific embodiment of the present application, the method for constructing the device for preparing cell clusters includes:

A1、采用光刻工艺制作具有柱状微腔阵列的聚甲基丙烯酸甲酯模板；A1. A polymethyl methacrylate template with a columnar microcavity array is fabricated by photolithography;

A2、利用模板法，将高温液态琼脂注入模板内，冷却凝固成型后从模板上剥离，得到可降解翻模***；A2. Using the template method, inject high-temperature liquid agar into the template, cool and solidify and peel it off the template to obtain a degradable stamp;

A3、将PDMS和丝素蛋白与交联剂混合，经消泡处理后(抽真空去除气泡)，覆盖在翻模***上，室温过夜，固化成型，得到PDMS和丝素蛋白混合基底；A3. Mix PDMS and silk fibroin with a cross-linking agent, and after defoaming treatment (vacuum to remove air bubbles), cover it on the stamp of the flip, overnight at room temperature, and solidify to form a mixed substrate of PDMS and silk fibroin;

A4、将翻模***通过加热融化，从PDMS和丝素蛋白混合基底上去除，得到具有微腔阵列的基底；A4. The stamp is melted by heating and removed from the mixed substrate of PDMS and silk fibroin to obtain a substrate with a microcavity array;

A5、外力挤压，将基底固定于12孔细胞培养板的孔内，灭菌处理。A5. Press external force to fix the substrate in the well of a 12-well cell culture plate, and sterilize it.

步骤A3中，PDMS、丝素蛋白和交联剂的质量比为5～10:0.6～60:1，所述交联剂为含氢硅油。In step A3, the mass ratio of PDMS, silk fibroin and cross-linking agent is 5-10:0.6-60:1, and the cross-linking agent is hydrogen-containing silicone oil.

在本申请的另一个具体实施方式中，用于制备细胞团簇的装置的构建方法包括：In another specific embodiment of the present application, the method for constructing the device for preparing cell clusters comprises:

B1、采用湿法蚀刻工艺制作具有倒锥形微腔阵列的硅片模板；B1. A silicon wafer template with an inverted tapered microcavity array is fabricated by a wet etching process;

B2、利用热压工艺，将硅片模板微图案面与聚甲基丙烯酸甲酯(PMMA)片材贴合、加热加压，在PMMA上得到微腔结构，机械剥离硅片模板和PMMA，得到不可降解翻模***；B2. Using the hot pressing process, the micropattern surface of the silicon wafer template is attached to the polymethyl methacrylate (PMMA) sheet, heated and pressurized to obtain a microcavity structure on the PMMA, and the silicon wafer template and PMMA are mechanically peeled off to obtain Non-degradable replica stamp;

B3、将PDMS和丝素蛋白与交联剂混合，经消泡处理后，覆盖在翻模***上，室温过夜，固化成型；B3. Mix PDMS and silk fibroin with a cross-linking agent, and after defoaming treatment, cover it on the stamp of the flip, and cure it overnight at room temperature;

B4、将翻模***通过机械力直接剥除，得到具有微腔阵列的基底；B4, directly peeling off the mold-reversing stamp by mechanical force to obtain a substrate with a microcavity array;

B5、外力挤压，将基底固定于12孔细胞培养板的孔内，灭菌处理。B5. Extrusion by external force, fixing the substrate in the well of the 12-well cell culture plate, and sterilizing.

步骤B3中，PDMS、丝素蛋白和交联剂的质量比为5～10:0.6～60:1，所述交联剂为含氢硅油。In step B3, the mass ratio of PDMS, silk fibroin and cross-linking agent is 5-10:0.6-60:1, and the cross-linking agent is hydrogen-containing silicone oil.

第三方面，本申请提供上述装置，或按照上述方法制备的装置在细胞团簇培养中的应用。In a third aspect, the present application provides the above device, or the application of the device prepared according to the above method in cell cluster culture.

本装置适合于：This device is suitable for:

1)培养单细胞团簇，如胰岛细胞、肝细胞培养成团簇；1) Cultivate single-cell clusters, such as islet cells and hepatocytes, into clusters;

2)干细胞培养和诱导分化，如胚胎干细胞、间充质干细胞成团簇培养并进一步诱导分化等；2) Stem cell culture and induced differentiation, such as embryonic stem cells and mesenchymal stem cells are cultured in clusters and further induced to differentiate, etc.;

3)多细胞共培养，如内皮细胞、成纤维细胞和肝实质细胞、胰岛细胞共培养，组织碎片与间充质干细胞成团共培养等。3) Multi-cell co-culture, such as endothelial cells, fibroblasts and hepatocytes, pancreatic islet cells co-culture, tissue fragments and mesenchymal stem cells co-culture in clusters, etc.

本装置适合培养的细胞可选自以下一种或多种细胞：各种来源的胚胎干细胞、多能干细胞、诱导性多能干细胞、各种器官来源的干细胞、各种器官来源的祖细胞、间充质干细胞、各种干细胞诱导分化得到的细胞、各种器官来源的成纤维细胞、各种器官来源的上皮细胞、各种器官来源的表皮细胞、各种器官来源的内皮细胞、各种器官来源的肌细胞、羊膜细胞、视锥细胞、神经细胞、血细胞、红细胞、白细胞、血小板、血管细胞、吞噬细胞、免疫细胞、淋巴细胞、嗜酸性粒细胞、嗜碱性粒细胞、浆细胞、肥大细胞、抗原呈递细胞、单核吞噬细胞***的细胞、黑色素细胞、软骨细胞、骨来源细胞、平滑肌细胞、骨骼肌细胞、心肌细胞、分泌细胞、脂肪细胞、纤毛细胞、胰腺细胞、肾细胞、肠粘膜细胞、肝细胞、肝来源的干细胞或祖细胞、肝巨噬细胞、枯否细胞、星状细胞、胆管上皮细胞、肝窦内皮细胞和其他各种组织和器官来源细胞，以及各种肿瘤细胞、各种用于免疫治疗的细胞、各种经过基因编辑、病毒包装或改造的细胞与细胞系。The cells suitable for culturing in this device can be selected from one or more of the following cells: embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, stem cells derived from various organs, progenitor cells derived from various organs, Mesenchymal stem cells, cells derived from various stem cells, fibroblasts derived from various organs, epithelial cells derived from various organs, epidermal cells derived from various organs, endothelial cells derived from various organs, and various organ sources muscle cells, amniotic cells, cone cells, nerve cells, blood cells, red blood cells, white blood cells, platelets, vascular cells, phagocytes, immune cells, lymphocytes, eosinophils, basophils, plasma cells, mast cells , antigen presenting cells, cells of the mononuclear phagocyte system, melanocytes, chondrocytes, bone-derived cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, secretory cells, adipocytes, ciliated cells, pancreatic cells, kidney cells, intestinal mucosa cells, hepatocytes, liver-derived stem or progenitor cells, hepatic macrophages, Kupffer cells, stellate cells, bile duct epithelial cells, hepatic sinusoidal endothelial cells and other cells of various tissue and organ origin, as well as various tumor cells, Various cells for immunotherapy, various gene-edited, virally packaged or engineered cells and cell lines.

所述细胞特别优选为干细胞，更优选为胚胎干细胞或间充质干细胞。The cells are particularly preferably stem cells, more preferably embryonic stem cells or mesenchymal stem cells.

本装置可通过简单操作收集团簇。例如可选择疏水性材料制备微腔阵列基底，或用表面活性剂处理基底材料，减弱细胞对基底的粘附，通过吹打的方式即可完成细胞团簇的脱离和收集，便于进行后续操作。This device can collect clusters by simple operation. For example, hydrophobic materials can be selected to prepare microcavity array substrates, or the substrate materials can be treated with surfactants to weaken the adhesion of cells to the substrates. The detachment and collection of cell clusters can be completed by pipetting, which is convenient for subsequent operations.

本装置可通过合适的基底保证细胞团簇的稳定生长，进行细胞团簇的长期培养和下游分化研究，并进一步应用于细胞治疗、高通量模型构建、类器官构建、药物筛选、组织再生和体外人工***等方面。The device can ensure the stable growth of cell clusters through a suitable substrate, conduct long-term culture and downstream differentiation studies of cell clusters, and be further applied to cell therapy, high-throughput model construction, organoid construction, drug screening, tissue regeneration and In vitro artificial systems, etc.

借由上述技术方案，本申请至少具有下列优点及有益效果：By the above technical solution, the present application has at least the following advantages and beneficial effects:

本申请提供的装置包括具有微腔阵列的基底和商用多孔培养板；每个微腔形成细胞团簇生长的空间，还可具备非垂直壁面，通过形状和体积调控获得的细胞团簇性质。本申请提供的装置可以便捷、大规模制备细胞团簇，可重复使用；可调节基底的弹性模量，以适应各种细胞、多种细胞共培养所要求的条件。采用本装置制备的细胞团簇活性高、大小均一、形状完整、性能可控、生物学性能良好，可用于细胞治疗、高通量模型构建、多细胞共培养、干细胞分化、类器官构建、药物筛选、组织再生和体外人工***等方面。The device provided in this application includes a substrate with a microcavity array and a commercial multi-well culture plate; each microcavity forms a space for the growth of cell clusters, and can also have non-vertical walls, and the properties of cell clusters obtained through shape and volume control. The device provided by the present application can prepare cell clusters conveniently and on a large scale, and can be used repeatedly; the elastic modulus of the substrate can be adjusted to adapt to the conditions required by the co-culture of various cells and various cells. The cell clusters prepared by this device have high activity, uniform size, complete shape, controllable performance and good biological performance, and can be used for cell therapy, high-throughput model construction, multi-cell co-culture, stem cell differentiation, organoid construction, drug Screening, tissue regeneration and in vitro artificial systems.

具体地，本申请提供的大规模细胞团簇制备装置具有可重复使用、非垂直壁面、快速大规模制备、物化性能可调控和满足高通量培养的特点。Specifically, the large-scale cell cluster preparation device provided by the present application has the characteristics of reusability, non-vertical wall surface, rapid large-scale preparation, regulated physicochemical properties, and high-throughput culture.

(一)本申请中的大规模细胞团簇制备装置可重复使用。本申请中的大规模细胞团簇制备装置经过清洗灭菌后重复使用，且能保持微图形精度和培养所需无菌环境。(1) The large-scale cell cluster preparation device in this application is reusable. The large-scale cell cluster preparation device in the present application can be reused after cleaning and sterilization, and can maintain the precision of the micropattern and the sterile environment required for cultivation.

(二)本装置可具备非垂直壁面。本装置可通过蚀刻技术得到的非垂直壁面模板制得，弥补目前利用光刻技术或一次模板法仅制得垂直壁面微腔的不足。(2) The device may have non-vertical walls. The device can be prepared by using a non-vertical wall template obtained by etching technology, which makes up for the shortage of only vertical wall microcavity prepared by photolithography technology or one-step template method.

(三)本装置可通过快速、可控、大规模的制备方法获得。本装置通过两步模板法制备得到微腔基底，避免微腔模板的阴阳模反转，可应用于复杂模具，也可以大规模制备获取。(3) The device can be obtained by a rapid, controllable and large-scale preparation method. The device prepares the micro-cavity substrate by a two-step template method, avoids the reversal of the male and female molds of the micro-cavity template, and can be applied to complex molds and can also be prepared and obtained on a large scale.

(四)本装置可根据需要调控其物化性能。可通过对细胞团簇尺寸、基底材料性能、机械强度等物化因素进行调控，从而实现细胞团簇的均质性生长、长期培养和下游研究，可用于不同细胞种类、不同细胞尺寸的培养、分化和功能维持，也可以完成细胞团簇的脱离和收集。(4) The device can adjust its physicochemical properties as required. By regulating the physical and chemical factors such as cell cluster size, substrate material properties, mechanical strength, etc., the homogeneous growth, long-term culture and downstream research of cell clusters can be realized. It can be used for the culture and differentiation of different cell types and different cell sizes. And function maintenance, the detachment and collection of cell clusters can also be completed.

(五)本装置可实现细胞团簇的高通量制备。本申请中的大规模细胞团簇制备装置借助计算机辅助设计和微图形化技术，可在同一模板、同一基底上实现各种微腔的形状和尺寸，从而实现同一培养条件下不同细胞团簇尺寸和分布的比较，对于通量研究和筛选具有重要意义。(5) The device can realize high-throughput preparation of cell clusters. The large-scale cell cluster preparation device in this application can realize the shape and size of various microcavities on the same template and the same substrate by means of computer-aided design and micro-patterning technology, so as to realize different cell cluster sizes under the same culture conditions. The comparison with distribution is of great significance for flux studies and screening.

附图说明Description of drawings

图1为本申请用于制备细胞团簇的装置及细胞团簇培养的示意图。FIG. 1 is a schematic diagram of the apparatus for preparing cell clusters and the culture of cell clusters in the present application.

图2为本申请用于制备细胞团簇的装置的不同微腔形状的示意图。其中，1)～4)为不同微腔形状。FIG. 2 is a schematic diagram of different microcavity shapes of the apparatus for preparing cell clusters of the present application. Wherein, 1) to 4) are different microcavity shapes.

图3为本申请装置制备时模板法过程的示意图。FIG. 3 is a schematic diagram of the template method process during the preparation of the device of the present application.

图4为本申请较佳实施例中脂肪干细胞在细胞团簇制备装置中的分化情况。其中，A是脂肪干细胞光镜图，B是分化后类胰岛细胞的光镜图，C是类胰岛细胞的免疫荧光染色图(scale bar＝200μm)。FIG. 4 shows the differentiation of adipose stem cells in the cell cluster preparation device according to the preferred embodiment of the present application. Among them, A is the light microscope image of adipose stem cells, B is the light microscope image of islet-like cells after differentiation, and C is the immunofluorescence staining image of islet-like cells (scale bar=200 μm).

图5为本申请较佳实施例中类胰岛细胞和内皮细胞在细胞团簇制备装置中的共培养情况。其中，A是类胰岛细胞和内皮细胞共培养的光镜图(scale bar＝500μm)，B是共培养细胞的免疫荧光染色图(scale bar＝200μm)。FIG. 5 shows the co-culture of islet-like cells and endothelial cells in the cell cluster preparation device according to the preferred embodiment of the present application. Among them, A is the light microscope image of the co-culture of islet cells and endothelial cells (scale bar=500 μm), and B is the immunofluorescence staining image of the co-cultured cells (scale bar=200 μm).

图6为本申请对比例中PDMS与丝素蛋白混合基底与纯PDMS基底在培养细胞方面的差异。FIG. 6 is the difference in culturing cells between the PDMS and silk fibroin mixed substrate and the pure PDMS substrate in the comparative example of the present application.

具体实施方式detailed description

本申请提供一种大规模制备细胞团簇的装置和方法，通过该装置和方法，可以获得微米级别的细胞/共培养细胞团簇，所制备的细胞/共培养细胞团簇具有活性高、大小均一、形状完整、性能可控、生物学性能良好的特点。本装置可重复使用，并可以采用合适的基底保证细胞团簇的稳定生长，从而进行长期培养和下游分化研究，此外，也可以根据需要完成细胞团簇的脱离和收集，以便后续工作。The present application provides a device and method for large-scale preparation of cell clusters. Through the device and method, micron-scale cell/co-culture cell clusters can be obtained, and the prepared cells/co-culture cell clusters have high activity and large size. It has the characteristics of uniformity, complete shape, controllable performance and good biological performance. The device is reusable, and a suitable substrate can be used to ensure the stable growth of cell clusters for long-term culture and downstream differentiation studies. In addition, the detachment and collection of cell clusters can also be completed as required for subsequent work.

本申请通过尺寸可调的微腔结构、刚度合适的基底一次性得到数以万计的活性高、大小均一、形状完整、性能可控、生物学性能良好的细胞/共培养细胞团簇，可满足细胞治疗、高通量模型构建、多细胞共培养、干细胞分化、类器官构建、药物筛选、组织再生和体外人工***等下游应用的多种要求。本申请提供的装置示意图见图1。In the present application, tens of thousands of cells/co-cultured cell clusters with high activity, uniform size, complete shape, controllable performance and good biological performance can be obtained at one time through a microcavity structure with adjustable size and a substrate with suitable stiffness. It meets various requirements for downstream applications such as cell therapy, high-throughput model construction, multi-cell co-culture, stem cell differentiation, organoid construction, drug screening, tissue regeneration, and in vitro artificial systems. The schematic diagram of the device provided in this application is shown in FIG. 1 .

本申请提供的细胞团簇制备装置，主要组成部分为具有微腔阵列的基底。The cell cluster preparation device provided in the present application is mainly composed of a substrate with a microcavity array.

本申请提供的微腔阵列基底的每个微腔是细胞生长的空间，其形状和体积影响细胞团簇的特性和生理功能。形状可为圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形和不规则形状等，，尤其以倒锥形这类非垂直壁面为优先项，微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，从而大致决定细胞团簇生长的尺寸和空间。微腔示意图可见图2。 Each microcavity of the microcavity array substrate provided by the present application is a space for cell growth, and its shape and volume affect the properties and physiological functions of the cell clusters. The shape can be cylindrical, square, rectangular, triangular, diamond, hexagonal, inverted cone and irregular shape, etc., especially the non-vertical wall surface such as inverted cone is the priority, The upper surface area of the microcavity is 0.04-1 mm ² , and the depth of the microcavity is 100-500 μm, which roughly determines the size and space of the cell cluster growth. The schematic diagram of the microcavity can be seen in Figure 2.

本申请提供的微腔阵列含有微腔数量1000～5000左右，视微腔尺寸与模板整体尺寸而定，阵列图案和尺寸可相同，也可不同，排列可整齐均一，也可自定义排列，同一基底阵列可包含不同形状、尺寸和排列方式的微腔。The microcavity array provided in this application contains about 1000 to 5000 microcavities, depending on the size of the microcavity and the overall size of the template. The array pattern and size can be the same or different, and the arrangement can be neat and uniform, or customized. Substrate arrays can contain microcavities of different shapes, sizes, and arrangements.

本申请提供的细胞团簇制备装置可配合商业常规多孔培养板(如6孔板、12孔板、24孔板等)使用。The cell cluster preparation device provided in this application can be used with commercial conventional multi-well culture plates (eg, 6-well plate, 12-well plate, 24-well plate, etc.).

本装置的微腔基底的弹性模量为0.1MPa～10MPa，可通过微腔基底材料的成分、浓度和固化特性调节弹性模量。The elastic modulus of the microcavity substrate of the device is 0.1 MPa to 10 MPa, and the elastic modulus can be adjusted by the composition, concentration and curing characteristics of the microcavity substrate material.

本申请提供上述细胞团簇制备装置的制备方式。装置制备方法包括以下步骤：The present application provides a preparation method of the above cell cluster preparation device. The device preparation method includes the following steps:

2)制备翻模***。对于液态或半固态***材料，利用模板法，将***材料覆盖在模板上，固化成型后从模板上剥离，得到翻模***。对于热塑性固态材料，利用热压法，将模具微图案与***材料(如聚甲基丙烯酸甲酯)贴合后加压、加热，再机械或化学剥离后，得到翻模***。2) Preparation of stamps. For the liquid or semi-solid stamp material, the stamp material is covered on the template by the template method, and then peeled off from the template after curing to obtain a re-molded stamp. For thermoplastic solid materials, using the hot pressing method, the mold micro-pattern and the stamp material (such as polymethyl methacrylate) are laminated, pressurized, heated, and then mechanically or chemically peeled off to obtain a stamped stamp.

3)将基底材料与交联剂混合(进行抽真空操作去除气泡)，浇铸在翻模***表面，进行消泡处理后固化成型；3) Mix the base material with the cross-linking agent (vacuuming to remove air bubbles), cast it on the surface of the stamp, and cure and form after defoaming;

5)将基底固定于细胞培养板的微孔内，高温灭菌或紫外灭菌处理；5) Fix the substrate in the micropores of the cell culture plate, and perform high temperature sterilization or ultraviolet sterilization;

6)可将多种细胞先后种植于微腔基底上，实现尺寸和生物性能可控的多细胞共培养细胞团簇的培养。6) A variety of cells can be successively planted on the microcavity substrate to realize the cultivation of multi-cell co-culture cell clusters with controllable size and biological properties.

装置制备过程示意图见图3。The schematic diagram of the device preparation process is shown in Figure 3.

所述微腔模板为自定义、具有微腔阵列、可重复使用的模板。本申请实施例中，所述微腔模板可选择已经商业化的产品，如底部具有400μm微孔阵列的多孔培养板Agreewell ^TM400(Stemcell)，也可以选择自定义模板，采用本领域公知的微图形化方法如干法蚀刻、湿法蚀刻、激光切割雕刻、微图形模具成型等结合计算机辅助设计自定义制备。 The microcavity template is a self-defined, reusable template with a microcavity array. In the examples of this application, the microcavity template can be selected from a commercialized product, such as the multi-well culture plate Agreewell ^™ 400 (Stemcell) with a 400 μm microwell array at the bottom, or a custom template can be selected, using a microchannel well-known in the art. Patterning methods such as dry etching, wet etching, laser cutting engraving, micro-pattern mold forming, etc. are custom-made in combination with computer-aided design.

微腔模板的材料可为硅、铝、铁、锡、玻璃、聚甲基丙烯酸甲酯、聚二甲基硅氧烷、聚己内酯、聚三亚甲基碳酸酯、聚四氟乙烯、聚氧化乙烯、聚乙烯醋酸乙烯酯、聚对二氧环己酮、聚醚醚酮等。The material of the microcavity template can be silicon, aluminum, iron, tin, glass, polymethyl methacrylate, polydimethylsiloxane, polycaprolactone, polytrimethylene carbonate, polytetrafluoroethylene, poly Ethylene oxide, polyethylene vinyl acetate, polydioxanone, polyether ether ketone, etc.

微腔模板可控调节微腔模板上微腔的形状、尺寸和数量，形状可为圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形和不规则形状等，尤其以倒锥形这类非垂直壁面为优先项，微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，数量1000～5000左右，视微腔尺寸与模板整体尺寸决定。微腔模板的形状和尺寸不必均一。 The microcavity template can controllably adjust the shape, size and quantity of the microcavity on the microcavity template, and the shape can be cylindrical, square column, rectangular column, triangular column, diamond column, hexagonal column, inverted cone and no Regular shape, etc., especially the non-vertical wall surface such as inverted cone is the priority, the upper surface area of the microcavity is 0.04～1mm ² , the depth of the microcavity is 100～500μm, and the number is about 1000～5000, depending on the size of the microcavity and the template Overall size is determined. The shape and size of the microcavity template need not be uniform.

微腔模板的整体尺寸和形状一般取决于配套使用的商业常规多孔培养板，一般为圆形，直径等于或略小于多孔培养板的孔径，厚度不决定最终基底厚度，以省材为主，一般为100μm～2cm不等。The overall size and shape of the micro-chamber template generally depends on the commercial conventional multi-well culture plate used. It is generally circular, with a diameter equal to or slightly smaller than the pore size of the multi-well culture plate. The thickness does not determine the final substrate thickness. It ranges from 100 μm to 2 cm.

所述翻模***为用于进行中间翻模的可降解材料或不可降解材料，通过模板法或热压法制得，其图案为模板微腔(阴模)的阳模，用于两步翻模、拓印到基底上制备微腔基底。The mold-turning stamp is a degradable or non-degradable material used for intermediate mold-turning, obtained by a template method or a hot-pressing method, and its pattern is the male mold of the template microcavity (female mold), which is used for two-step mold-turning. , rubbing onto the substrate to prepare the microcavity substrate.

翻模***的形状可为圆柱形、正方柱形、长方形柱、三角柱形、菱柱形、六棱柱形、倒锥形和不规则形状等，多为凸出结构，结构上表面面积为0.04～1mm ²，高度为100～500μm，数量1000～5000左右，形状和尺寸不必均一。 The shapes of the stamps can be cylindrical, square, rectangular, triangular, diamond, hexagonal, inverted cone and irregular, etc. Most of them are protruding structures, and the upper surface area of the structure is 0.04~ 1 mm ² , the height is 100 to 500 μm, the number is about 1000 to 5000, and the shape and size are not necessarily uniform.

用于翻模***的可降解材料可为明胶、明胶衍生物、琼脂、琼脂糖、

F-127、聚乙烯醇、聚乙二醇等牺牲材料，便于后续制得基底后通过加热、水溶等牺牲方式去除。用于翻模***的不可降解材料可为聚甲基丙烯酸甲酯、环氧树脂、酚醛树脂、聚氯乙烯树脂、不饱和聚酯树脂、石膏、硅胶等中的至少一种。 The degradable materials used for the stamp can be gelatin, gelatin derivatives, agar, agarose,

Sacrificial materials such as F-127, polyvinyl alcohol, polyethylene glycol, etc., are convenient to be removed by sacrificial methods such as heating and water solubility after the subsequent preparation of the substrate. The non-degradable material used for the stamping can be at least one of polymethyl methacrylate, epoxy resin, phenolic resin, polyvinyl chloride resin, unsaturated polyester resin, gypsum, silica gel, and the like.

所述翻模***材料的质量百分比浓度为0.1％～80％，优选为1％～25％。The mass percentage concentration of the stamp material is 0.1% to 80%, preferably 1% to 25%.

所述微腔基底为培养细胞团簇的基底。微腔基底可用的材料为聚丙烯、聚苯乙烯、聚丙烯酰胺、聚乳酸、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、聚氧化乙烯、丝素蛋白、丝素蛋白衍生物、壳聚糖、明胶、明胶衍生物、藻酸盐、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白和纤维蛋白等中的至少一种，优选聚二甲基硅氧烷和丝素蛋白的混合物，其中聚二甲基硅氧烷和丝素蛋白的质量比为10:1～1:10。The microcavity substrate is the substrate for culturing cell clusters. Available materials for microcavity substrates are polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd, polydimethylsiloxane, polyanhydride, polyester, polyamide, Polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, silk fibroin, silk fibroin derivative Compounds, Chitosan, Gelatin, Gelatin Derivatives, Alginate, Agar, Matrigel, Collagen, Collagen Derivatives, Hyaluronic Acid, Hyaluronic Acid Derivatives, Cellulose, Cellulose-Derived Materials, Proteoglycans, Proteoglycans At least one of derivatives, glycoproteins, glycoprotein-derived materials, laminin, fibronectin, and fibrin, etc., preferably a mixture of polydimethylsiloxane and silk fibroin, wherein polydimethylsiloxane The mass ratio of alkane and silk fibroin is 10:1～1:10.

所述基底材料的质量百分比浓度可以是0.1％～80％，优选1％～25％。The mass percentage concentration of the base material may be 0.1% to 80%, preferably 1% to 25%.

微腔基底的成型固定是利用翻模***两步翻模。将未成型固定的基底材料覆盖在翻模***上，通过真空消泡、静置等方式保证基底材料与翻模***紧密贴合，然后根据基底材料和翻模***材料的特性采取交联剂固化、热固化、光固化、紫外固化、pH调节固化等方式完成基底材料的成型固定，得到具有等同于微腔模板的微腔基底。The forming and fixing of the micro-cavity substrate is a two-step overmold using the overmold stamp. Cover the unmolded and fixed base material on the stamp, and ensure that the base material is closely attached to the stamp by vacuum defoaming, standing, etc., and then use a cross-linking agent to cure according to the characteristics of the base material and stamp material. , thermal curing, light curing, UV curing, pH adjustment curing and other methods to complete the forming and fixing of the base material, and obtain a microcavity substrate equivalent to a microcavity template.

微腔基底为保证细胞团簇的稳定生长，可以根据需要添加有利于细胞团簇粘附和生长的细胞外基质成分，如胶原、基质胶、蛋白多糖、糖蛋白、透明质酸、层连接蛋白或纤维连接蛋白。In order to ensure the stable growth of cell clusters, extracellular matrix components that are conducive to the adhesion and growth of cell clusters, such as collagen, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin, can be added to the microcavity substrate as needed. or fibronectin.

所述细胞外基质材料的质量百分比浓度为0.1％～80％，优选为1％～25％。The mass percentage concentration of the extracellular matrix material is 0.1%-80%, preferably 1%-25%.

微腔基底的弹性模量为0.1MPa～100MPa，通过基底材料的成分、浓度和固化特性调节。基底材料的成分和浓度可根据培养细胞团簇的特性进行选择，交联剂固化的材料可调节固化剂比例，热固化的材料可调节固化时间和固化温度，光固化或紫外固化的材料可调节光照强度和光照时间，从而调节弹性模量，一般而言，固化剂比例越大、固化温度越高、固化时间越长、光照强度越大，得到的基底弹性模量越大。The elastic modulus of the microcavity substrate is 0.1 MPa to 100 MPa, which is adjusted by the composition, concentration and curing characteristics of the substrate material. The composition and concentration of the base material can be selected according to the characteristics of the cultured cell clusters. The ratio of curing agent can be adjusted for materials cured by cross-linking agent. The curing time and curing temperature can be adjusted for thermally cured materials. In general, the greater the proportion of curing agent, the higher the curing temperature, the longer the curing time, and the greater the light intensity, the greater the elastic modulus of the obtained substrate.

所用交联剂可为含氢硅油、硅烷偶联剂、二价阳离子、京尼平、戊二醛、已二酸二酰肼、环氧氯丙烷、碳化二亚胺、凝血酶及其衍生物等中的至少一种，优选含氢硅油。The cross-linking agent used can be hydrogen-containing silicone oil, silane coupling agent, divalent cation, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide, thrombin and its derivatives At least one of such, preferably hydrogen-containing silicone oil.

所用交联剂的浓度为0.1mM～10M，优选1mM～100mM。The concentration of the cross-linking agent used is 0.1 mM to 10 M, preferably 1 mM to 100 mM.

所述基底材料与交联剂溶液按1000:1～1:1000的体积比混合，优选10:1～1:10。The base material and the crosslinking agent solution are mixed in a volume ratio of 1000:1 to 1:1000, preferably 10:1 to 1:10.

所述基底材料的热固化温度为10～100℃。The thermal curing temperature of the base material is 10-100°C.

所述基底材料的光照强度为0.5～1000lx。The light intensity of the base material is 0.5-1000 lx.

所述基底材料的pH值为3～12。The pH value of the base material is 3-12.

可降解翻模***在微腔基底成型固定后需要根据其材料特性选择牺牲去除。翻模***材料为温敏材料，如明胶、明胶衍生物、琼脂、琼脂糖等高温溶解材料则可通过加热去除，如

F-127等低温溶解材料可采用冷冻去除，材料为聚乙烯醇、聚乙二醇等水溶性材料可通过水溶方式去除。 After the microcavity substrate is molded and fixed, the biodegradable mold stamp needs to be sacrificed and removed according to its material properties. The stamp material is temperature-sensitive, such as gelatin, gelatin derivatives, agar, agarose and other high-temperature dissolving materials can be removed by heating, such as

Low-temperature dissolving materials such as F-127 can be removed by freezing, and water-soluble materials such as polyvinyl alcohol and polyethylene glycol can be removed by water-dissolving.

不可降解翻模***在微腔基底成型固定后机械剥离即可。The non-degradable mold stamp can be mechanically peeled off after the microcavity substrate is formed and fixed.

所述具有微腔阵列基底通过两步翻模制得，具备与上述微腔模板相同的微腔阵列，形状可为圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形和不规则形状等，尤其以倒锥形这类非垂直壁面为优先项，微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，数量1000～5000左右，形状和尺寸不必均一。 The substrate with microcavity array is obtained by two-step overmolding, and has the same microcavity array as the above-mentioned microcavity template, and the shape can be cylindrical, square column, rectangular column, triangular column, diamond column, six Prismatic, inverted cone and irregular shapes, etc., especially the non-vertical wall such as inverted cone is the priority, the upper surface area of the microcavity is 0.04～1mm ² , the depth of the microcavity is 100～500μm, and the number is 1000～5000 Left and right, shape and size need not be uniform.

微腔基底具有一定阵列的微腔，每个微腔的形状和体积构成细胞团簇生长的物理空间，与种植的细胞数量共同决定细胞团簇的尺寸，进而影响细胞团簇的功能表达。The microcavity substrate has a certain array of microcavities. The shape and volume of each microcavity constitute the physical space for the growth of cell clusters. Together with the number of cells to be planted, the size of the cell clusters is determined, which in turn affects the functional expression of the cell clusters.

所述具有微腔阵列的基底与常规细胞培养板粘着构成整体，构建方法包括但不局限于以下两种：The substrate with the microcavity array is adhered to the conventional cell culture plate to form a whole, and the construction methods include but are not limited to the following two:

1、设计和制备出需要尺寸和形状的微腔基底后，裁剪为合适形状，并嵌入商业常规多孔培养板中，利用压力和微腔基底材料的粘附性，将微腔基底与多孔培养皿表面连接形成整体，待灭菌处理后可即时使用或保存待用。1. After designing and preparing the microcavity substrate of the required size and shape, cut it into an appropriate shape, and embed it in a commercial conventional multi-well culture plate. Using the pressure and the adhesion of the microcavity substrate material, the microcavity substrate is connected to the multi-well culture dish. The surface is connected to form a whole, which can be used immediately after sterilization or stored for later use.

2、将微图形基底材料和翻模***同时置于商业常规多孔培养板，待微图形基底材料成型后去除翻模***，即得到成为整体的微图形基底-多用培养板，待灭菌处理后可即时使用或保存待用。2. Place the micro-pattern base material and the mold-turning stamp on a commercial conventional multi-hole culture plate at the same time, and remove the mold-turning stamp after the micro-pattern base material is formed, to obtain an integrated micro-pattern base-multi-purpose culture plate. After sterilization treatment Can be used immediately or saved for later use.

以下实施例用于说明本申请，但不用来限制本申请的范围。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。The following examples are used to illustrate the present application, but are not intended to limit the scope of the present application. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

聚甲基丙烯酸甲酯购自淘宝电商百邦亚克力加工店，平均分子量约200万道尔顿。Polymethyl methacrylate was purchased from Taobao e-commerce Baibang acrylic processing store, with an average molecular weight of about 2 million Daltons.

聚二甲基硅氧烷(PDMS)购自Dow Corning公司，货号7450507，为双组份硅橡胶，混合前黏度5500cps(25℃)，混合后黏度3900cps(25℃)。Polydimethylsiloxane (PDMS) was purchased from Dow Corning Company, item number 7450507, which is a two-component silicone rubber with a viscosity of 5500cps (25°C) before mixing and a viscosity of 3900cps (25°C) after mixing.

基本组分主要为二甲基硅油和铂系催化剂。The basic components are mainly dimethyl silicone oil and platinum-based catalyst.

固化剂主要为含氢硅油。The curing agent is mainly hydrogen-containing silicone oil.

丝素蛋白购自Sigma-Aldrich公司，货号5154，平均分子量为100KDa。Silk fibroin was purchased from Sigma-Aldrich Company, item number 5154, with an average molecular weight of 100KDa.

实施例1 用可降解翻模***制作细胞团簇制备装置的方法Example 1 The method of making a cell cluster preparation device with a degradable stamp

1、制备微腔模板1. Preparation of microcavity template

微腔模板采用湿法蚀刻制备的具有倒锥形微腔阵列的硅片，制备整体直径为3cm的圆形模板，模板上为含1500个微腔的倒锥形阵列，相邻微腔之间的距离为200μm，微腔为上表面为正方形的倒锥形，上表面面积为0.25mm ²，深度为500μm。 The microcavity template is a silicon wafer with an inverted tapered microcavity array prepared by wet etching, and a circular template with an overall diameter of 3 cm is prepared. The template is an inverted tapered array containing 1500 microcavities. The distance is 200 μm, the microcavity is an inverted cone with a square upper surface, the upper surface area is 0.25 mm ² , and the depth is 500 μm.

2、制备可降解翻模***2. Preparation of degradable stamps

翻模***采用琼脂材料。采用浓度为5％的琼脂溶液，高温高压溶解，趁热灌注于微腔模板中，每次灌注2ml琼脂即可，并在琼脂溶液凝固前用真空去除气泡。待琼脂冷却凝固后小心取出琼脂***。此处完成12个***的制备。The imprinted stamp is made of agar material. Agar solution with a concentration of 5% was used, dissolved at high temperature and high pressure, and poured into the micro-cavity template while still hot, with 2ml of agar per pour, and vacuum was used to remove air bubbles before the agar solution solidified. After the agar has cooled and solidified, carefully remove the agar stamp. The preparation of 12 stamps is done here.

3、制备微腔基底3. Preparation of the microcavity substrate

按质量比5：1将Sylgard 184 silicone elastomer kit(Dow Corning公司，货号7450507)中的PDMS基本组分(主要成分为二甲基硅油和铂系催化剂)与固化剂(主要成分为含氢硅油)充分混合，每克PDMS再加入100mg丝素蛋白，充分搅拌均匀，并进行真空消泡处理。将制备的12个琼脂***置于60mm直径培养皿中，具有微图案的表面朝上，将PDMS-丝素蛋白混合物浇铸在***表面，室温过夜。According to the mass ratio of 5:1, the basic components of PDMS (the main components are dimethyl silicone oil and platinum-based catalyst) and the curing agent (the main components are hydrogen-containing silicone oil) in the Sylgard 184 silicone elastomer kit (Dow Corning Company, product number 7450507) Mix well, add 100 mg of silk fibroin per gram of PDMS, stir well, and perform vacuum defoaming treatment. The prepared 12 agar stamps were placed in a 60 mm diameter petri dish with the micropatterned surface facing up, and the PDMS-silk fibroin mixture was cast on the stamp surface overnight at room temperature.

待PDMS和丝素蛋白混合物完全固化后，加热牺牲琼脂，得到含有12个具有微腔阵列的基底。利用打孔器，按照常规12孔培养皿的孔径大小取出PDMS和丝素蛋白混合物中含微腔阵列的部分，并根据需要裁减为合适厚度。得到的微腔基底大小均一，含有相同的微腔阵列。将微腔基底嵌入12孔培养皿中，利用压力和PDMS本身的粘附性，将微腔基底与12孔培养皿表面连接在一起。After the PDMS and silk fibroin mixture was completely solidified, the sacrificial agar was heated to obtain a substrate containing 12 microcavity arrays. Using a hole puncher, take out the part containing the microcavity array in the PDMS and silk fibroin mixture according to the pore size of a conventional 12-well petri dish, and cut it to an appropriate thickness as needed. The resulting microcavity substrates are uniform in size and contain identical microcavity arrays. The microcavity base was embedded in a 12-well petri dish, and the microcavity base was connected with the surface of the 12-well petri dish by using the pressure and the adhesion of PDMS itself.

最后对PDMS进行高温高压灭菌处理，处理后保持疏水状态。高压灭菌的条件为：105℃，0.15MPa灭菌30min。Finally, the PDMS was subjected to high temperature and high pressure sterilization treatment, and the hydrophobic state was maintained after treatment. The conditions of autoclaving are: 105°C, 0.15MPa for 30min.

所制备装置的形态、结构及性质如下：The morphology, structure and properties of the prepared device are as follows:

常规12孔培养皿底部为PDMS和丝素蛋白组成的微腔基底，基底整体为直径3cm的圆形，基底表面为微腔阵列，微腔形状为倒四方锥，上表面面积为0.25mm ²，深度为500μm，阵列排布密度为214个微孔/cm ²，相邻微腔之间的距离为200μm，基底弹性模量为5MPa，pH值为7.4。 The bottom of a conventional 12-well petri dish is a microcavity base composed of PDMS and silk fibroin, the whole base is a circle with a diameter of 3cm, the base surface is a microcavity array, the shape of the microcavity is an inverted square pyramid, and the upper surface area is 0.25mm ² . The depth is 500 μm, the array density is 214 micropores/cm ² , the distance between adjacent microcavities is 200 μm, the substrate elastic modulus is 5 MPa, and the pH value is 7.4.

实施例2 用不可降解翻模***制作细胞团簇制备装置的方法Example 2 The method of making a cell cluster preparation device with a non-degradable stamp

1、制备微腔模板1. Preparation of microcavity template

2、制备不可降解翻模***2. Preparation of non-degradable overmolded stamps

不可降解翻模***采用聚甲基丙烯酸甲酯(PMMA)。利用热压工艺，将硅片模板微图案面与聚甲基丙烯酸甲酯(PMMA)片材贴合，加热至130℃、加压至1MPa、保持30s，退火，在PMMA上得到微腔结构。机械剥离硅片模板和PMMA，得到不可降解翻模***。此处完成12个***的制备，此***可重复使用。The non-degradable overmolded stamp is made of polymethyl methacrylate (PMMA). Using the hot pressing process, the micropatterned surface of the silicon wafer template is bonded to a polymethyl methacrylate (PMMA) sheet, heated to 130 °C, pressurized to 1 MPa, held for 30 s, and annealed to obtain a microcavity structure on the PMMA. The silicon wafer template and PMMA were mechanically peeled off to obtain a non-degradable overmolded stamp. The preparation of 12 stamps is completed here, and this stamp can be reused.

3、制备微腔基底3. Preparation of the microcavity substrate

按质量比5:1将Sylgard 184 silicone elastomer kit中的PDMS基本组分(主要成分为二甲基硅油和铂系催化剂)与固化剂(主要成分为含氢硅油)充分混合，每克PDMS再加入100mg丝素蛋白，充分搅拌均匀，并进行真空消泡处理。将制备的12个PMMA***置于60mm直径培养皿中，具有微图案的表面朝上，将PDMS-丝素蛋白混合物浇铸在***表面，室温过夜。The basic components of PDMS in Sylgard 184 silicone elastomer kit (the main components are dimethyl silicone oil and platinum catalyst) and the curing agent (the main components are hydrogen-containing silicone oil) in the Sylgard 184 silicone elastomer kit are thoroughly mixed in a mass ratio of 5:1, and then add to each gram of PDMS. 100 mg of silk fibroin, fully stirred, and vacuum defoamed. The prepared 12 PMMA stamps were placed in a 60mm diameter petri dish with the micropatterned surface facing up, and the PDMS-silk fibroin mixture was cast on the stamp surface overnight at room temperature.

待PDMS和丝素蛋白混合物完全固化后，机械剥离PMMA***和PDMS-丝素蛋白基底，得到含有12个具有微腔阵列的基底。利用打孔器，按照常规12孔培养皿的孔径大小取出PDMS和丝素蛋白混合物中含微腔阵列的部分，并根据需要裁减为合适厚度。得到的微腔基底大小均一，含有相同的微腔阵列。将微腔基底嵌入12孔培养皿中，利用压力和PDMS本身的粘附性，将微腔基底与12孔培养皿表面连接在一起。After the PDMS and silk fibroin mixture was completely solidified, the PMMA stamp and the PDMS-silk fibroin substrate were mechanically peeled off to obtain a substrate containing 12 microcavity arrays. Using a hole puncher, take out the part containing the microcavity array in the PDMS and silk fibroin mixture according to the pore size of a conventional 12-well petri dish, and cut it to an appropriate thickness as needed. The resulting microcavity substrates are uniform in size and contain identical microcavity arrays. The microcavity base was embedded in a 12-well petri dish, and the microcavity base was connected with the surface of the 12-well petri dish by using the pressure and the adhesion of PDMS itself.

最后对装置进行高温高压灭菌处理，处理后保持疏水状态。高压灭菌的条件为：105℃，0.15MPa灭菌30min。高温高压灭菌不会破坏基底的空间结构。***用酒精清洗后晾干，以便之后使用。Finally, the device is subjected to high temperature and high pressure sterilization treatment, and the hydrophobic state is maintained after treatment. The conditions of autoclaving are: 105°C, 0.15MPa for 30min. Autoclaving does not destroy the spatial structure of the substrate. The stamp is rinsed with alcohol and dried for later use.

实施例3 利用细胞团簇制备装置诱导脂肪干细胞分化为胰岛细胞团Example 3 Using a cell cluster preparation device to induce adipose stem cells to differentiate into islet cell clusters

脂肪干细胞-类胰岛细胞分化液的配制：DMEM培养基和DMEM/F-12培养基按2:1体积比混合，加入Nicotinamide(5mM)，Activin A(4nM)，Exendin-4(20nM)，Pentagastrin(20nM)，hepatocyte growth factor(200pM)，B-27 supplement(1％)，N-2 supplement(1％)，双抗(1％)。Preparation of adipose stem cell-islet cell differentiation medium: DMEM medium and DMEM/F-12 medium were mixed in a 2:1 volume ratio, Nicotinamide (5mM), Activin A (4nM), Exendin-4 (20nM), Pentagastrin were added (20nM), hepatocyte growth factor (200pM), B-27 supplement (1%), N-2 supplement (1%), double antibody (1%).

本申请在现有脂肪干细胞-类胰岛细胞分化液的基础上进行了改良，增加了Activin A、Exendin-4、Pentagastrin、hepatocyte growth factor这四类促进胰腺分化的生长因子的浓度，并且降低了B-27 supplement、N-2 supplement这两类维持干细胞干性的培养基成分，提升了脂肪干细胞向胰腺细胞分化的效率。This application improves the existing adipose stem cell-islet cell differentiation solution, increases the concentration of four types of growth factors that promote pancreatic differentiation, Activin A, Exendin-4, Pentagastrin, and hepatocyte growth factor, and reduces B -27 supplement and N-2 supplement, two types of medium components that maintain stem cell stemness, improve the efficiency of adipose-derived stem cells to differentiate into pancreatic cells.

1、准备台盼蓝染色液：将160μl分化液和20μl台盼蓝溶液混合备用。1. Prepare trypan blue staining solution: mix 160 μl differentiation solution and 20 μl trypan blue solution for use.

2、取2瓶T75的脂肪干细胞(ATCC)，对于每瓶细胞，消化细胞；终止消化，离心。2. Take 2 flasks of T75 adipose stem cells (ATCC), for each flask of cells, digest the cells; terminate the digestion, and centrifuge.

3、2支离心管吸走上清，分别加入1ml分化液吹打重悬；将所有细胞悬液汇聚到一支离心管中。3. Aspirate the supernatant from 2 centrifuge tubes, add 1ml of differentiation medium to resuspend, and pool all cell suspensions into one centrifuge tube.

4、吹打使细胞尽可能分散，取20μl细胞悬液至台盼蓝染液中，室温染色2min。4. To disperse the cells as much as possible by pipetting, take 20 μl of the cell suspension into trypan blue staining solution, and stain at room temperature for 2 min.

5、对这200μl细胞悬液，吹打使细胞尽可能分散；取20μl加入细胞计数板，进行细胞计数。根据计数结果，将2ml细胞悬液调节至活细胞浓度为9×10 ⁵个/ml。 5. To the 200μl cell suspension, pipetting to disperse the cells as much as possible; add 20μl to the cell counting plate and count the cells. According to the counting results, 2 ml of the cell suspension was adjusted to a viable cell concentration of 9×10 ⁵ cells/ml.

6、向实施例1制备的12孔细胞团簇制备装置中每孔加入2ml已调节密度的细胞悬液，最终得到细胞密度为1.8×10 ⁶个/孔，每个微孔约含有1200个细胞；吹打分散细胞，注意避免产生气泡。 6. Add 2ml of cell suspension with adjusted density to each well of the 12-well cell cluster preparation device prepared in Example 1, and finally obtain a cell density of 1.8×10 ⁶ cells/well, and each microwell contains about 1200 cells ; Disperse cells by pipetting, taking care to avoid air bubbles.

7、用配平板进行离心操作，200g离心3min；显微镜下观察，确保每个微腔有细胞。7. Centrifuge at 200g for 3 min with the matching plate; observe under the microscope to ensure that there are cells in each microchamber.

8、将细胞团簇制备装置放入培养箱，共分化6天左右，每3天进行半换液。8. Put the cell cluster preparation device into the incubator for about 6 days of differentiation, and half-change the medium every 3 days.

9、观察脂肪干细胞分化。光学显微镜下观察脂肪干细胞向类胰岛细胞的分化情况。吹打收集细胞，可获得大量细胞团簇，不易分散，边缘清晰。9. Observe the differentiation of adipose stem cells. The differentiation of adipose stem cells into islet-like cells was observed under a light microscope. To collect cells by pipetting, a large number of cell clusters can be obtained, which are not easy to disperse and have clear edges.

脂肪干细胞在实施例1的细胞团簇制备装置中的分化情况见图4。其中，A是脂肪干细胞的形貌光镜图；B是脂肪干细胞在细胞团簇制备装置中分化6天后的形貌光镜图，光镜下可以观察到细胞从松散分布到紧密堆积，形成细胞团簇，边缘清晰、尺寸均一、形状完整；The differentiation of adipose stem cells in the cell cluster preparation device of Example 1 is shown in Figure 4 . Among them, A is the morphological light microscope image of adipose stem cells; B is the morphological light microscope image of adipose stem cells after 6 days of differentiation in the cell cluster preparation device. Clusters with clear edges, uniform size and complete shape;

10、检测脂肪干细胞分化情况。为了检测装置中脂肪干细胞向类胰岛细胞的分化情况，采用免疫荧光染色检测胰岛β细胞的特异性标记蛋白的表达(如Pdx1)。10. Detect the differentiation of adipose stem cells. In order to detect the differentiation of adipose stem cells into islet-like cells in the device, immunofluorescence staining was used to detect the expression of specific marker proteins (such as Pdx1) of islet beta cells.

收集细胞：收集装置中每个孔的上清液；每个孔加入1ml PBS吹打一次，收集液体，并再重复该过程1次。转移至50ml离心管，1000rpm3min离心，弃上清，除去大部分单细胞，并置于培养皿中。Harvest cells: collect the supernatant from each well in the device; add 1 ml of PBS to each well by pipetting once, collect the liquid, and repeat the process 1 more time. Transfer to a 50ml centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, remove most of the single cells, and place it in a petri dish.

免疫荧光染色：用磷酸缓冲液(PBS)洗涤细胞团簇；4％多聚甲醛在室温下固定30分钟，后用PBS洗涤1次，5分钟；含0.3％Triton-X(Sigma，X100)冰上透化20分钟，后用PBS洗涤1次，5分钟；5％牛血清白蛋白(Multicell，800-096-EG)封闭1小时，后用PBS洗涤1次，5分钟；加入按说明书稀释的一抗，Pdx1(Abcam，ab47383)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa

594(abcam，ab150080)，室温避光孵育2小时，后用PBS洗涤3次，每次5分钟；加入DAPI染细胞核，室温避光孵育10分钟。用激光共聚焦显微镜(LSCM,Nikon，Z2)观察记录。 Immunofluorescence staining: Cell clusters were washed with phosphate buffered saline (PBS); fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then washed once with PBS for 5 minutes; with 0.3% Triton-X (Sigma, X100) on ice Permeabilized for 20 minutes, then washed once with PBS for 5 minutes; blocked with 5% bovine serum albumin (Multicell, 800-096-EG) for 1 hour, washed once with PBS for 5 minutes; The primary antibody, Pdx1 (Abeam, ab47383), was incubated at 4°C overnight. Wash 3 times with PBS, 5 minutes each time; add the corresponding secondary antibody Alexa

594 (abcam, ab150080), incubated at room temperature for 2 hours in the dark, and then washed with PBS for 3 times, 5 minutes each time; DAPI was added to stain the nuclei, and the cells were incubated in the dark at room temperature for 10 minutes. Recordings were observed with a laser confocal microscope (LSCM, Nikon, Z2).

实验结果见图4，其中C是类胰岛细胞的免疫荧光染色图，分化后的细胞团是Pdx1阳性表达，证明脂肪干细胞分化成功，得到的类胰岛细胞团表达胰岛β细胞的一定功能。The experimental results are shown in Figure 4, in which C is the immunofluorescence staining of islet-like cells. The differentiated cell mass is positive for Pdx1, which proves that the adipose stem cells are successfully differentiated, and the obtained islet-like cell mass expresses a certain function of islet β cells.

本实施例中采用微腔阵列进行细胞培养，为准3D环境，与平面培养相比，具有如下技术优势：培养周期更短，可在2天后即形成细胞团簇；成团效率更高，接近100％，几乎不产生单细胞；分化效率更高，近80％细胞表达胰岛相关蛋白；细胞团簇获取高效便捷，避免消化等操作，减少细胞团簇的松散和凋亡；细胞团簇尺寸一致性良好，且能够保证细胞团簇的生物性能。In this example, the microcavity array is used for cell culture, which is a quasi-3D environment. Compared with flat culture, it has the following technical advantages: the culture period is shorter, and cell clusters can be formed after 2 days; the clustering efficiency is higher, close to 100%, almost no single cells are produced; the differentiation efficiency is higher, nearly 80% of cells express islet-related proteins; cell clusters are obtained efficiently and conveniently, avoiding operations such as digestion, and reducing the loosening and apoptosis of cell clusters; the size of cell clusters is consistent It has good properties and can guarantee the biological properties of cell clusters.

与悬浮培养相比，技术优势主要在于：脂肪干细胞贴壁能力强，难以悬浮培养，可用本申请装置构建准3D环境；成团效率更高，几乎不产生单细胞；分化效率更高；细胞团簇更紧密，细胞间接触更充分；细胞团簇尺寸一致性良好，保证细胞团簇的生物性能。Compared with suspension culture, the main technical advantages are: adipose stem cells have strong adherence ability and are difficult to be cultured in suspension, and the device of the present application can be used to build a quasi-3D environment; the clustering efficiency is higher, almost no single cells are produced; the differentiation efficiency is higher; cell clusters The clusters are more compact and the contact between cells is more sufficient; the size of the cell clusters is consistent, which ensures the biological properties of the cell clusters.

实施例4 利用细胞团簇制备装置共培养类胰岛细胞与内皮细胞Example 4 Co-cultivation of islet cells and endothelial cells using a cell cluster preparation device

1、类胰岛细胞的准备1. Preparation of islet-like cells

由脂肪干细胞分化得到，分化方法类似于实施例3。脂肪干细胞在补充有10％FBS、1％双抗的DMEM培养液中培养，并由脂肪干细胞-类胰岛细胞分化液分化为类胰岛细胞。It is obtained by differentiation of adipose stem cells, and the differentiation method is similar to that of Example 3. The adipose stem cells were cultured in DMEM medium supplemented with 10% FBS and 1% double antibody, and differentiated into islet-like cells from the adipose stem cell-islet-like cell differentiation medium.

脂肪干细胞-类胰岛细胞分化液的配制：DMEM培养基和DMEM/F-12培养基按2:1混合，加入Nicotinamide(5mM)，Activin A(4nM)，Exendin-4(20nM)，Pentagastrin(20nM)，hepatocyte growth factor(200pM)，B-27 supplement(1％)，N-2 supplement(1％)，双抗(1％)。Preparation of adipose stem cell-islet cell differentiation medium: DMEM medium and DMEM/F-12 medium were mixed at a ratio of 2:1, Nicotinamide (5mM), Activin A (4nM), Exendin-4 (20nM), Pentagastrin (20nM) were added ), hepatocyte growth factor (200pM), B-27 supplement (1%), N-2 supplement (1%), double antibody (1%).

2、内皮细胞的准备2. Preparation of endothelial cells

内皮细胞(PUMC-HUVEC-T1，北京协和细胞资源中心)在添加有10％FBS、1％双抗(青霉素-链霉素)的DMEM培养液中培养。Endothelial cells (PUMC-HUVEC-T1, Peking Union Cell Resource Center) were cultured in DMEM medium supplemented with 10% FBS and 1% double antibody (penicillin-streptomycin).

3、细胞团簇共培养3. Cell cluster co-culture

共培养液：胰岛细胞分化液和内皮细胞液按1：1体积比混合。Co-culture medium: Islet cell differentiation medium and endothelial cell fluid were mixed in a volume ratio of 1:1.

1)准备台盼蓝染色液：配制160μl培养液+20μl台盼蓝溶液混合备用。1) Prepare trypan blue staining solution: prepare 160 μl of culture medium + 20 μl of trypan blue solution and mix for later use.

2)取1瓶T75的内皮细胞和1瓶T75的脂肪干细胞，消化细胞；终止消化，离心。2) Take 1 bottle of T75 endothelial cells and 1 bottle of T75 adipose stem cells, digest the cells; terminate the digestion, and centrifuge.

3)2支离心管吸走上清，各加入1ml共培养液吹打重悬、汇总，使细胞尽可能分散，内皮细胞和脂肪干细胞各取20μl细胞悬液至台盼蓝染液中，室温染色2min。取20μl加入细胞计数板，进行细胞计数。根据计数结果，用共培养液将两种细胞的细胞悬液调节至活细胞浓度为9×10 ⁵个/ml，然后1：1混合，得到细胞悬液中含内皮细胞和脂肪干细胞各4.5×10 ⁵个/ml。 3) Aspirate the supernatant from 2 centrifuge tubes, add 1 ml of co-culture solution to each, resuspend and aggregate, so that the cells are dispersed as much as possible. Take 20 μl of the cell suspension for endothelial cells and adipose stem cells into trypan blue staining solution, and stain at room temperature. 2min. Add 20 μl to a cell counting plate for cell counting. According to the counting results, the cell suspensions of the two types of cells were adjusted to a viable cell concentration of 9×10 ⁵ cells/ml with the co-culture medium, and then mixed 1:1 to obtain 4.5× endothelial cells and adipose stem cells in the cell suspension. 10 ⁵ / ml.

4)向实施例2制备的12孔细胞团簇培养装置中加入2ml细胞悬液，每个孔最终有细胞1.8×10 ⁶个/孔，每个微腔约含有1200个细胞；吹打分散细胞，注意避免产生气泡。 4) Add 2 ml of cell suspension to the 12-well cell cluster culture device prepared in Example 2, each well finally has 1.8×10 ⁶ cells/well, and each microchamber contains about 1200 cells; pipetting to disperse the cells, Take care to avoid air bubbles.

5)用配平板进行离心操作，200g离心3min；显微镜下观察，确保每个微孔有细胞。5) Centrifuge at 200 g for 3 min with a matching plate; observe under a microscope to ensure that there are cells in each microwell.

6)将细胞团簇制备装置放入培养箱，共分化6天左右，每3天进行半换液。6) Put the cell cluster preparation device into the incubator for about 6 days of differentiation, and half-change the medium every 3 days.

4、细胞团簇检测4. Cell cluster detection

1)观察细胞团簇共培养情况。1) Observe the co-culture of cell clusters.

光学显微镜下观察类胰岛细胞和内皮细胞的共培养情况。吹打收集细胞，可获得大量细胞团簇，不易分散，边缘清晰。The co-culture of islet cells and endothelial cells was observed under a light microscope. To collect cells by pipetting, a large number of cell clusters can be obtained, which are not easy to disperse and have clear edges.

类胰岛细胞和内皮细胞在实施例2的细胞团簇制备装置中的共培养情况见图5。其中，A是类胰岛细胞和内皮细胞共培养的形貌光镜图，可看到松散细胞逐渐聚集，形成紧密团簇形态，边缘清晰，细胞团簇大小均一。Figure 5 shows the co-culture of islet cells and endothelial cells in the cell cluster preparation device of Example 2. Among them, A is the morphological light microscope image of islet-like cells and endothelial cells co-cultured. It can be seen that loose cells gradually aggregate to form tight clusters with clear edges and uniform size of cell clusters.

2)检测共培养情况下类胰岛细胞和内皮细胞功能。2) Detect the function of islet-like cells and endothelial cells in co-culture.

为了检测共培养情况下类胰岛细胞和内皮细胞的功能表达，采用免疫荧光染色检测胰岛β细胞的特异性标记蛋白的表达(如Pdx1)和内皮细胞的特异性标记蛋白的表达(如CD31)。In order to detect the functional expression of islet-like cells and endothelial cells in co-culture, immunofluorescence staining was used to detect the expression of islet β-cell-specific marker proteins (such as Pdx1) and endothelial cell-specific marker proteins (such as CD31).

收集细胞：收集装置中每个孔的上清液；每个孔加入1ml PBS吹打一次，收集液体，并再重复该过程1次。转移至50ml离心管，1000rpm离心3min，弃上清，除去大部分单细胞，并置于培养皿中。Harvest cells: collect the supernatant from each well in the device; add 1 ml of PBS to each well by pipetting once, collect the liquid, and repeat the process 1 more time. Transfer to a 50ml centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, remove most of the single cells, and place it in a petri dish.

免疫荧光染色：用磷酸缓冲液(PBS)洗涤细胞团簇；4％多聚甲醛在室温下固定30分钟，后用PBS洗涤1次，5分钟；含0.3％Triton-X(Sigma，X100)冰上透化20分钟，后用PBS洗涤1次，5分钟；5％牛血清白蛋白(Multicell，800-096-EG)封闭1小时，后用PBS洗涤1次，5分钟；加入按说明书稀释的一抗，Pdx1(Abcam，ab47383)和CD31(Abcam，ab24590)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa

594(abcam，ab150080)和Alexa

488(abcam，ab150113)，室温避光孵育2小时，后用PBS洗涤3次，每次5分钟；加入DAPI染细胞核，室温避光孵育10分钟。用激光共聚焦显微镜(LSCM,Nikon，Z2)观察记录。 Immunofluorescence staining: Cell clusters were washed with phosphate buffered saline (PBS); fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then washed once with PBS for 5 minutes; with 0.3% Triton-X (Sigma, X100) on ice Permeabilized for 20 minutes, then washed once with PBS for 5 minutes; blocked with 5% bovine serum albumin (Multicell, 800-096-EG) for 1 hour, washed once with PBS for 5 minutes; Primary antibodies, Pdx1 (Abeam, ab47383) and CD31 (Abeam, ab24590), were incubated overnight at 4°C. Wash 3 times with PBS, 5 minutes each time; add the corresponding secondary antibody Alexa

594 (abcam, ab150080) and Alexa

488 (abcam, ab150113), incubated at room temperature for 2 hours in the dark, and then washed with PBS for 3 times, 5 minutes each time; DAPI was added to stain the nuclei, and the cells were incubated in the dark at room temperature for 10 minutes. Recordings were observed with a laser confocal microscope (LSCM, Nikon, Z2).

类胰岛细胞和内皮细胞在实施例2的细胞团簇制备装置中的共培养情况见图5。其中，B是共培养细胞的免疫荧光染色图，细胞团簇中类胰岛细胞呈Pdx1阳性表达，内皮细胞呈CD31阳性表达，证明在细胞团簇制备中类胰岛细胞和内皮细胞能够维持分化功能表达。Figure 5 shows the co-culture of islet cells and endothelial cells in the cell cluster preparation device of Example 2. Among them, B is the immunofluorescence staining of the co-cultured cells, the islet cells in the cell cluster are Pdx1 positive, and the endothelial cells are CD31 positive, which proves that the islet cells and endothelial cells can maintain the expression of differentiation function in the cell cluster preparation .

本实施例中采用微腔阵列进行细胞培养，为准3D环境，与平面培养相比，具有如下技术优势：培养周期更短，可在2天后即形成细胞团簇；成团效率更高，接近100％，几乎不产生单细胞；分化效率更高，近80％细胞表达胰岛相关蛋白；可加强不同细胞之间的接触，达到共培养的目的；细胞团簇获取高效便捷，避免消化等操作，减少细胞团簇的松散和凋亡；细胞团簇尺寸一致性良好，且能够保证细胞团簇的生物性能。In this example, the microcavity array is used for cell culture, which is a quasi-3D environment. Compared with flat culture, it has the following technical advantages: the culture period is shorter, and cell clusters can be formed after 2 days; the clustering efficiency is higher, close to 100%, almost no single cells are produced; the differentiation efficiency is higher, nearly 80% of cells express islet-related proteins; the contact between different cells can be strengthened to achieve the purpose of co-culture; cell clusters are obtained efficiently and conveniently, avoiding operations such as digestion, Reduce the loosening and apoptosis of cell clusters; the size of cell clusters is consistent, and the biological properties of cell clusters can be guaranteed.

对比例：Comparative ratio:

实施例1和实施例2中都采用了PDMS与丝素蛋白混合物作为微腔基底。PDMS是常用材料，可作为弹性基底用于细胞培养等领域。本申请将PDMS与丝素蛋白的混合物作为基底材料，取得了显著的技术效果：In both Example 1 and Example 2, a mixture of PDMS and silk fibroin was used as the microcavity substrate. PDMS is a common material that can be used as an elastic substrate for cell culture and other fields. This application uses the mixture of PDMS and silk fibroin as the base material, and has achieved remarkable technical effects:

1、丝素蛋白具有良好的生物相容性，可用于细胞培养基质，与PDMS混合作为基底后，可调节细胞生长情况，其纳米级多孔结构加强了氧气交换和渗透作用，更有利于细胞快速增殖。PDMS与丝素蛋白混合基底与纯PDMS基底在培养细胞方面的差异见图6，对应于实施例3，可看出PDMS与丝素蛋白混合基底可促进细胞快速增殖。1. Silk fibroin has good biocompatibility and can be used as a cell culture medium. After mixing with PDMS as a substrate, it can regulate cell growth. Its nano-scale porous structure enhances oxygen exchange and penetration, which is more conducive to rapid cell growth. proliferation. The difference between the PDMS and silk fibroin mixed substrate and the pure PDMS substrate in terms of culturing cells is shown in Figure 6, which corresponds to Example 3. It can be seen that the PDMS and silk fibroin mixed substrate can promote rapid cell proliferation.

2、丝素蛋白具有疏水性，作为混合基底，有利于细胞从基底脱落，方便无损收集细胞。纯PDMS作为基底材料制备本申请的装置，细胞团簇的回收率为85％，采用PDMS与丝素蛋白的混合体系作为基底材料制备本申请的装置，细胞团簇的回收率可达94％。2. Silk fibroin has hydrophobicity. As a mixed substrate, it is conducive to the shedding of cells from the substrate and facilitates the collection of cells without damage. Pure PDMS is used as the base material to prepare the device of the present application, and the recovery rate of cell clusters is 85%. The mixed system of PDMS and silk fibroin is used as the base material to prepare the device of the present application, and the recovery rate of cell clusters can reach 94%.

虽然，上文中已经用一般性说明及具体实施方案对本申请作了详尽的描述，但在本申请基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本申请精神的基础上所做的这些修改或改进，均属于本申请要求保护的范围。Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application fall within the scope of protection claimed in the present application.

Claims

用于制备细胞团簇的装置，其特征在于，所述装置包括细胞培养板以及固定于细胞培养板的孔内且具有微腔阵列的基底；A device for preparing cell clusters, characterized in that the device comprises a cell culture plate and a substrate fixed in a hole of the cell culture plate and having a microcavity array;

每个微腔构成细胞团簇生长的空间；微腔的形状选自圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形或不规则形状，尤其以倒锥形这类非垂直壁面为优先项；微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，基底上排列的微腔数量为1000～5000个。 Each microcavity constitutes a space for the growth of cell clusters; the shape of the microcavity is selected from cylindrical, square, rectangular, triangular, rhomboid, hexagonal, inverted cone or irregular shapes, especially The non-vertical wall surface such as inverted cone is the priority; the upper surface area of the microcavity is 0.04-1 mm ² , the depth of the micro-cavity is 100-500 μm, and the number of micro-cavities arranged on the substrate is 1000-5000.
根据权利要求1所述的装置，其特征在于，用于制作基底的材料选自聚丙烯、聚苯乙烯、聚丙烯酰胺、聚乳酸、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、聚氧化乙烯、丝素蛋白、丝素蛋白衍生物、壳聚糖、明胶、明胶衍生物、藻酸盐、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白和纤维蛋白中的至少一种，优选聚二甲基硅氧烷和丝素蛋白的混合物；和/或The device according to claim 1, wherein the material used for making the substrate is selected from the group consisting of polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethyl Silicone, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate Ester, polyethylene oxide, silk fibroin, silk fibroin derivatives, chitosan, gelatin, gelatin derivatives, alginate, agar, matrigel, collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives , at least one of cellulose, cellulose-derived materials, proteoglycans, proteoglycan derivatives, glycoproteins, glycoprotein-derived materials, laminin, fibronectin, and fibrin, preferably polydimethylsiloxane and a mixture of silk fibroin; and/or

所述细胞培养板为商用多孔培养板，优选6孔板、12孔板或24孔板。The cell culture plate is a commercial multi-well culture plate, preferably a 6-well plate, a 12-well plate or a 24-well plate.
根据权利要求1所述的装置，其特征在于，所述基底的弹性模量为0.1MPa～10MPa；和/或The device according to claim 1, wherein the elastic modulus of the substrate is 0.1 MPa to 10 MPa; and/or

所述基底的pH值为3～12；和/或The substrate has a pH of 3 to 12; and/or

所述基底的厚度为100μm～2cm。The thickness of the substrate is 100 μm˜2 cm.
根据权利要求1-3任一项所述的装置，其特征在于，所述基底还包被有细胞外基质成分；The device according to any one of claims 1-3, wherein the substrate is further coated with extracellular matrix components;

优选地，所述细胞外基质成分选自胶原、基质胶、蛋白多糖、糖蛋白、透明质酸、层连接蛋白、纤维连接蛋白中的至少一种。Preferably, the extracellular matrix component is selected from at least one of collagen, matrigel, proteoglycan, glycoprotein, hyaluronic acid, laminin, and fibronectin.
用于制备细胞团簇的装置的构建方法，其特征在于，包括：A method for constructing a device for preparing cell clusters, characterized in that it comprises:

1)采用干法蚀刻、湿法蚀刻或光刻工艺制作具有微腔阵列的模板；1) Use dry etching, wet etching or photolithography to make a template with a microcavity array;

2)制备翻模***。对于液态或半固态的***材料，利用模板法，将***材料覆盖在模板上，固化成型后从模板上剥离，得到翻模***。对于热塑性固态材料，利用热压法，将模具微图案与***材料贴合后加压、加热，再机械或化学剥离后，得到翻模***。2) Preparation of stamps. For the liquid or semi-solid stamp material, the stamp material is covered on the template by using the template method, and then peeled off from the template after curing and forming, to obtain the over-molded stamp. For thermoplastic solid materials, using the hot pressing method, the mold micro-pattern and the stamp material are laminated, pressurized and heated, and then mechanically or chemically peeled off to obtain a re-molded stamp.

3)将基底材料与交联剂混合，消泡处理后浇铸在翻模***表面，固化成型；3) Mix the base material with the cross-linking agent, cast it on the surface of the mold-turning stamp after defoaming treatment, and cure and form;

4)将翻模***从上述成型材料中机械剥离，或者通过化学或物理方法从上述成型材料中去除翻模***，得到具有微腔阵列的基底；4) mechanically peeling off the mold-reversing seal from the above-mentioned molding material, or removing the mold-reversing seal from the above-mentioned molding material by chemical or physical methods to obtain a substrate with a microcavity array;

5)将基底固定于细胞培养板的微孔内，灭菌处理；5) Fix the substrate in the microwell of the cell culture plate, and sterilize it;

步骤3)中所述的基底材料选自聚丙烯、聚苯乙烯、聚丙烯酰胺、聚乳酸、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、聚氧化乙烯、丝素蛋白、丝素蛋白衍生物、壳聚糖、明胶、明胶衍生物、藻酸盐、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白和纤维蛋白中的至少一种，优选聚二甲基硅氧烷和丝素蛋白的混合物；The base material described in step 3) is selected from polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester , polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, silk fibroin, Silk Fibroin Derivatives, Chitosan, Gelatin, Gelatin Derivatives, Alginate, Agar, Matrigel, Collagen, Collagen Derivatives, Hyaluronic Acid, Hyaluronic Acid Derivatives, Cellulose, Cellulose-Derived Materials, Protein at least one of polysaccharides, proteoglycan derivatives, glycoproteins, glycoprotein-derived materials, laminin, fibronectin and fibrin, preferably a mixture of polydimethylsiloxane and silk fibroin;

步骤1)中所述模板上的微腔的形状选自圆柱形、正方柱形、长方柱形、三角柱形、菱柱形、六棱柱形、倒锥形或不规则形状，尤其以倒锥形这类非垂直壁面为优先项；微腔的上表面面积为0.04～1mm ²，微腔深度为100～500μm，模板上排列的微腔的数量为1000～5000个。 The shape of the microcavity on the template in step 1) is selected from cylindrical, square, rectangular, triangular, rhombic, hexagonal, inverted cone or irregular shapes, especially inverted cones. The non-vertical wall surface such as shape is the priority; the upper surface area of the microcavity is 0.04-1 mm ² , the depth of the micro-cavity is 100-500 μm, and the number of micro-cavities arranged on the template is 1000-5000.
根据权利要求5所述的方法，其特征在于，步骤1)中用于制作模板的材料选自硅、铝、铁、锡、玻璃、聚甲基丙烯酸甲酯、聚二甲基硅氧烷、聚己内酯、聚三亚甲基碳酸酯、聚四氟乙烯、聚氧化乙烯、聚乙烯醋酸乙烯酯、聚对二氧环己酮、聚醚醚酮中的至少一种；和/或The method according to claim 5, wherein in step 1), the material used for making the template is selected from the group consisting of silicon, aluminum, iron, tin, glass, polymethyl methacrylate, polydimethylsiloxane, At least one of polycaprolactone, polytrimethylene carbonate, polytetrafluoroethylene, polyethylene oxide, polyethylene vinyl acetate, polydioxanone, polyether ether ketone; and/or

步骤2)中所述的***材料为可降解材料或不可降解材料，所述可降解材料选自明胶、明胶衍生物、琼脂、琼脂糖、
F-127、聚乙烯醇、聚乙二醇中的至少一种，所述不可降解材料选自聚甲基丙烯酸甲酯、环氧树脂、酚醛树脂、聚氯乙烯树脂、不饱和聚酯树脂、石膏、硅胶中的至少一种。 The seal material described in step 2) is a degradable material or a non-degradable material, and the degradable material is selected from gelatin, gelatin derivatives, agar, agarose,
At least one of F-127, polyvinyl alcohol, polyethylene glycol, the non-degradable material is selected from polymethyl methacrylate, epoxy resin, phenolic resin, polyvinyl chloride resin, unsaturated polyester resin, At least one of gypsum and silica gel.
根据权利要求6所述的方法，其特征在于，步骤2)中所述翻模材料的质量百分比浓度为0.1％～80％，优选1％～25％；和/或The method according to claim 6, characterized in that, the mass percentage concentration of the overmolding material in step 2) is 0.1% to 80%, preferably 1% to 25%; and/or

步骤3)中所述交联剂的浓度为0.1mM～10M，优选1mM～100mM；和/或The concentration of the cross-linking agent in step 3) is 0.1mM～10M, preferably 1mM～100mM; and/or

所述交联剂选自含氢硅油、硅烷偶联剂、二价阳离子、京尼平、戊二醛、已二酸二酰肼、环氧氯丙烷、碳化二亚胺、凝血酶及其衍生物中的至少一种，优选含氢硅油；和/或The crosslinking agent is selected from the group consisting of hydrogen-containing silicone oil, silane coupling agent, divalent cation, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide, thrombin and derivatives thereof. At least one of them, preferably hydrogen-containing silicone oil; and/or

步骤3)中所述基底材料与交联剂按1000:1～1:1000的体积比混合，优选10:1～1:10；和/或In step 3), the base material and the cross-linking agent are mixed in a volume ratio of 1000:1 to 1:1000, preferably 10:1 to 1:10; and/or

步骤3)中固化的条件为：温度10～100℃和/或光照强度0.5～1000lx。The curing conditions in step 3) are: temperature 10-100° C. and/or light intensity 0.5-1000 lx.
根据权利要求5-7任一项所述的方法，其特征在于，包括：The method according to any one of claims 5-7, characterized in that, comprising:

A1、采用光刻工艺制作具有柱状微腔阵列的聚甲基丙烯酸甲酯模板；A1. A polymethyl methacrylate template with a columnar microcavity array is fabricated by photolithography;

A2、利用模板法，将高温液态琼脂注入模板内，冷却凝固成型后从模板上剥离，得到可降解翻模***；A2. Using the template method, inject high-temperature liquid agar into the template, cool and solidify and peel it off the template to obtain a degradable stamp;

A3、将聚二甲基硅氧烷和丝素蛋白与交联剂混合，经消泡处理后，覆盖在翻模***上，室温过夜，固化成型，得到聚二甲基硅氧烷和丝素蛋白混合基底；A3. Mix the polydimethylsiloxane and silk fibroin with a cross-linking agent, and after defoaming treatment, cover it on the stamp of the flip, overnight at room temperature, and solidify to obtain polydimethylsiloxane and silk fibroin. protein mix base;

A4、将翻模***通过加热融化，从聚二甲基硅氧烷和丝素蛋白混合基底上去除，得到具有微腔阵列的基底；A4. Melt the stamp by heating and remove it from the mixed substrate of polydimethylsiloxane and silk fibroin to obtain a substrate with a microcavity array;

A5、外力挤压，将基底固定于12孔细胞培养板的孔内，灭菌处理；A5. Press external force to fix the substrate in the well of a 12-well cell culture plate, and sterilize it;

步骤A3中，聚二甲基硅氧烷、丝素蛋白和交联剂的质量比为5～10：0.6～60:1，所述交联剂为含氢硅油。In step A3, the mass ratio of polydimethylsiloxane, silk fibroin and cross-linking agent is 5-10:0.6-60:1, and the cross-linking agent is hydrogen-containing silicone oil.
根据权利要求5-7任一项所述的方法，其特征在于，包括：The method according to any one of claims 5-7, characterized in that, comprising:

B1、采用湿法刻蚀工艺制作具有倒锥形微腔阵列的硅片模板；B1. A silicon wafer template with an inverted tapered microcavity array is fabricated by a wet etching process;

B2、利用热压工艺，将硅片模板微图案面与聚甲基丙烯酸甲酯(PMMA)片材贴合、加热加压，在PMMA上得到微腔结构，机械剥离硅片模板和PMMA，得到不可降解翻模***；B2. Using the hot pressing process, the micropattern surface of the silicon wafer template is attached to the polymethyl methacrylate (PMMA) sheet, heated and pressurized to obtain a microcavity structure on the PMMA, and the silicon wafer template and PMMA are mechanically peeled off to obtain Non-degradable replica stamp;

B3、将聚二甲基硅氧烷和丝素蛋白与交联剂混合，经消泡处理后，覆盖在翻模***上，室温过夜，固化成型；B3. Mix the polydimethylsiloxane and silk fibroin with the cross-linking agent, and after defoaming treatment, cover it on the mold-turning stamp, and cure it overnight at room temperature;

B4、将翻模***通过机械力直接剥除，得到具有微腔阵列的基底；B4, directly peeling off the mold-reversing stamp by mechanical force to obtain a substrate with a microcavity array;

B5、外力挤压，将基底固定于12孔细胞培养板的孔内，灭菌处理；B5. Extrusion by external force, fixing the substrate in the well of the 12-well cell culture plate, and sterilizing;

步骤B3中，聚二甲基硅氧烷、丝素蛋白和交联剂的质量比为5～10：0.6～60:1，所述交联剂为含氢硅油。In step B3, the mass ratio of polydimethylsiloxane, silk fibroin and cross-linking agent is 5-10:0.6-60:1, and the cross-linking agent is hydrogen-containing silicone oil.
权利要求1-4任一项所述装置，或按照权利要求5-9任一项所述方法制备的装置在细胞团簇培养中的应用。The application of the device according to any one of claims 1 to 4, or the device prepared according to the method according to any one of claims 5 to 9, in cell cluster culture.