WO2013075392A1

WO2013075392A1 - Method and device for constructing three-dimensional cellular microenvironment on the basis of transparent sponge scaffold

Info

Publication number: WO2013075392A1
Application number: PCT/CN2012/001013
Authority: WO
Inventors: 杜亚楠; 赵姗
Original assignee: 清华大学
Priority date: 2011-11-23
Filing date: 2012-07-27
Publication date: 2013-05-30
Also published as: CN103131625B; CN103131625A; CN104053459B; CN104053459A

Abstract

Provided are a method and a device for constructing a three-dimensional cellular microenvironment. The method comprises the following steps: 1) establishing a three-dimensional transparent sponge scaffold or a three-dimensional transparent sponge scaffold array; 2) establishing a thin sample liquid layer containing molecules, a material, cells, and a mixture thereof; 3) combining the transparent sponge scaffold or transparent sponge scaffold array in 1) with the thin liquid layer in 2), and completing loading of a sample liquid, thereby achieving construction of a three-dimensional microenvironment. The method and device provide a simple and practical platform for research on the growth, proliferation, label-free observation, and functional analysis of cells in a three-dimensional microenvironment in the fields such as the biomedical research and the drug research and development, and rapidly and losslessly realize the patterning of the three-dimensional microenvironment of molecules, a material, cells, and a mixture thereof, high flux synchronous loading, and label-free real-time observation.

Description

基于透明海绵支架构建三维细胞微环境的方法及装置技术领域 Method and device for constructing three-dimensional cell microenvironment based on transparent sponge scaffold

本发明涉及一种构建三维微环境的方法及装置，特别涉及一种与传统两维细胞培养技术同样简易方便操作的方法（easy as 2D) 实现包括可溶性因子、生物材料和细胞的三维细胞微环境的构建的方法和装置，属于生物医学工程领域。 The invention relates to a method and a device for constructing a three-dimensional microenvironment, in particular to a method as simple and convenient to operate as the traditional two-dimensional cell culture technology (easy as 2D), realizing a three-dimensional cell microenvironment including soluble factors, biological materials and cells. The method and device for construction belong to the field of biomedical engineering.

背景技术 Background technique

两维细胞培养（基于商业化的培养皿或多孔板）技术发展已有百余年的历史，在生命科学基础研究、制药产业、医学研究等领域具有广泛应用。但在很多情况下，两维细胞培养环境与细胞在体内生长的真实环境相距甚远。因此这种简化的两维微环境不能很好的模拟和重现体内的三维微环境。而依赖于动物体内实验的研究过程复杂且与人体反应具有差异性。因此,三维细胞培养技术在近年来得到了大幅度的发展。 Two-dimensional cell culture (based on commercial petri dishes or multi-well plates) has been developed for more than 100 years and is widely used in life science basic research, pharmaceutical industry, medical research and other fields. However, in many cases, the two-dimensional cell culture environment is far removed from the real environment in which cells grow in the body. Therefore, this simplified two-dimensional microenvironment does not simulate and reproduce the three-dimensional microenvironment in the body. The research process that relies on in vivo experiments in animals is complex and different from human response. Therefore, three-dimensional cell culture technology has been greatly developed in recent years.

三维细胞培养技术是指将不同种类的细胞种植到具有三维结构的材料载体中，使细胞能够在载体的三维立体空间结构中迁移、生长、行使功能。该技术的目的是模拟体内细胞生长环境，其核心因素是细胞与三维微环境之间的相互作用，即细胞与分子、细胞与细胞外基质（Extracellular matrix, ECM) 以及细胞与细胞的相互作用。三维细胞微环境较真实地在体外模拟和重现体内细胞生长环境与状态（生长、分化、极化、细胞间相互作用等），细胞在基因表达、基质分泌及细胞功能活动等方面与两维培养均有明显差异，作为生理模型研究细胞对于药物反应有较准确的预测性，同样作为病理模型具有很高的仿生性。因此在体外构建三维细胞微环境实现分子、材料和细胞在三维水平上相互作用的研究对于加速生物、医学和药物开发等领域的发展具有重要意义。 The three-dimensional cell culture technique refers to planting different kinds of cells into a material carrier having a three-dimensional structure, so that the cells can migrate, grow, and function in the three-dimensional spatial structure of the carrier. The goal of this technique is to mimic the cell growth environment in vivo. The core factor is the interaction between cells and the three-dimensional microenvironment, namely the interaction between cells and molecules, cells and extracellular matrix (ECM), and cells and cells. The three-dimensional cell microenvironment simulates and reproduces the cell growth environment and state (growth, differentiation, polarization, cell-cell interaction, etc.) in vitro, and the cells are in two dimensions in terms of gene expression, matrix secretion, and cell function. There are significant differences in culture. As a physiological model, cells have a more accurate predictive effect on drug response, and also have high biomimeticity as a pathological model. Therefore, the study of constructing a three-dimensional cellular microenvironment in vitro to achieve interaction between molecules, materials and cells at a three-dimensional level is of great significance for accelerating the development of fields such as biology, medicine and drug development.

目前，细胞三维培养方式主要有两种：水凝胶（hydrogel)培养方式和支架（scaffold) 培养方式。水凝胶培养方式是将细胞和材料的悬浮液在一定条件下交联成水凝胶，细胞在水凝胶的交联网络体系内实现三维培养。常用的成胶（gelling)方式有：温度转变（如 collagen、 matrigel) 、 H转变（如 chitosan) 、添加离子（如 alginate) 、光暴露（如 hyaluronic acid or dextran-containing vinyl groups)等等。支架培养方式是指将细胞或细胞 -材料的悬浮液种植到已经成型的三维支架材料中实现三维培养。目前种植的方法分为两大类：静态种植和动态种植。静态种植一般将细胞或细胞-材料的悬浮液直接滴加在支架上；动态种植借助外部动力（如旋转种植、表面超声波种植、离心种植、磁场种植等）使细胞较高效、均匀地渗入支架内部。与传统两维细胞培养方式相比，水凝胶培养体系中，细胞在成胶过程中需要同预聚物一起经历交联过程，不可避免受到伤害；且水凝胶体系中约 90%成分为水，机械性能较差，不适合长期换液增殖培养及定量检测。但水凝胶具有良好的光学性能，可以像两维培养方式一样在普通白光显微镜下对细胞生长状态进行实时无标记观测，在血管新生、肿瘤发生学等领域具有广泛应用。支架培养方式中，静态种植法简便、使用广泛，但也最低效，动态种植法具有外部机械力损伤细胞的潜在危险，且支架培养体系光学性能较差，细胞在支架体系内的分布、生长、迁移等变化无法实时观测，必须借助荧光标记、固定染色等手段，无疑增大了实验的复杂性和技术难度。但支架具有良好的机械性能，可以像两维培养方式一样对细胞进行长期换液增殖培养，在组织工程、临床检测等领域具有广泛应用。 At present, there are two main methods for three-dimensional cell culture: hydrogel culture method and scaffold culture method. The hydrogel culture method is to cross-link a suspension of cells and materials into a hydrogel under certain conditions, and the cells are three-dimensionally cultured in a hydrogel crosslinked network system. Commonly used gelling methods are: temperature transitions (such as collage, matrigel), H transitions (such as chitosan), addition of ions (such as alginate), light exposure (such as hyaluronic acid or dextran-containing vinyl groups), and the like. The stent culture method refers to planting a cell or a suspension of cells-material into a three-dimensional scaffold material that has been formed to realize three-dimensional culture. Current methods of planting fall into two broad categories: static planting and dynamic planting. Static planting generally drops cell or cell-material suspension directly onto the scaffold; dynamic planting uses external power (such as rotary planting, surface ultrasonic implant, centrifugal planting, magnetic field planting, etc.) to allow cells to penetrate the stent more efficiently and evenly. . Compared with the traditional two-dimensional cell culture method, in the gelation culture system, the cells need to undergo a crosslinking process together with the prepolymer during the gelation process, which is inevitably damaged; and about 90% of the components in the hydrogel system are Water, poor mechanical properties, not suitable for long-term fluid exchange culture and quantitative detection. However, hydrogels have good optical properties and can be used for real-time unlabeled observation of cell growth under ordinary white light microscopy in the same way as two-dimensional culture. It has wide applications in angiogenesis and oncogenesis. In the stent culture method, the static implantation method is simple and widely used, but it is also the least effective. The dynamic implantation method has the potential danger of damage to cells by external mechanical force, and the optical performance of the stent culture system is poor, the distribution and growth of cells in the stent system, Changes such as migration cannot be observed in real time, and it is necessary to use fluorescence labeling, fixed staining, etc., which undoubtedly increases the complexity and technical difficulty of the experiment. However, the stent has good mechanical properties and can be used for long-term fluid-changing and proliferating culture of cells as in the two-dimensional culture method. Nutrient, widely used in tissue engineering, clinical testing and other fields.

近期，三维细胞培养技术发展迅速，市场上逐渐推出十余种三维细胞培养产品（如 Alvetex、 AlgiMatrix GEM、 Microtissues、 RAFT、 n3D 等），均产自美国、英国、瑞士等地。产品主要分为两种类型：水凝胶型和支架型。例如 qgelbio公司推出的 QGel™, 是一种 PEG粉末，将其与细胞悬浮液和 QGel^TMbuffer混合，以 QGel™Disc Caster为模具，制备载有细胞的三维水凝胶薄片；而 3Dbioteck公司推出的 3D Insert™-PS和 3D Insert™-PCL, 是一种基于多孔板的内嵌式多孔支架，细胞悬浮液直接滴加在内部连通的多孔支架上达到三维培养的目的。两种类型产品相比较，水凝胶型三维细胞培养方式前期制备过程较复杂，需要多步操作，细胞和预聚物一起交联成胶有损伤风险，且需要配套模具，价格较高，但由于水凝胶光学性能较好，后续实验中可用显微镜在白光下观察细胞状态；而多孔支架型三维细胞培养方式操作过程简便，技术难度低，但由于多孔支架光学性能差，无法在普通白光显微镜下直接观察细胞状态，实验中需要对细胞进行荧光标记和荧光成像。根据 DDW (Drug Discovery World) 调查统计，目前仅有 7%科研人员逐渐将两维细胞培养方式转向三维培养方式， 7%人员持期望和积极态度，而 86% 人员拒绝使用，主要原因是他们要求三维细胞研究手段要和传统的两维细胞研究手段在整套实验体系中保持一致：培养方式上无需特殊工具和技巧，检测方式上可依赖于常规设备（off-the-shelf instruments ) 和方法进行实时检测。 Recently, three-dimensional cell culture technology has developed rapidly. More than ten kinds of three-dimensional cell culture products (such as Alvetex, AlgiMatrix GEM, Microtissues, RAFT, n3D, etc.) have been introduced in the market, all from the United States, Britain, Switzerland and other places. There are two main types of products: hydrogel type and stent type. E.g. qgelbio company launched QGel ™, PEG is a powder, it is mixed with the cell suspension and QGel ^TM buffer, QGel ™ Disc Caster in a mold, three-dimensional hydrogel preparation containing a sheet of cells; the company launched 3Dbioteck 3D InsertTM-PS and 3D InsertTM-PCL are in-line porous scaffolds based on multi-well plates. The cell suspension is directly added to the interconnected porous scaffold for three-dimensional culture. Compared with the two types of products, the pre-preparation process of the hydrogel-type three-dimensional cell culture method is complicated, and requires multiple steps. The cells and the prepolymer are crosslinked into a gel, which has the risk of damage, and requires a matching mold, and the price is high, but Since the hydrogel has good optical properties, the microscope can be used to observe the cell state under white light in the subsequent experiments; while the porous scaffold type three-dimensional cell culture mode has a simple operation process and low technical difficulty, but the optical performance of the porous scaffold is poor, and it cannot be used in ordinary white light microscopy. The cell state is directly observed, and the cells are required to be fluorescently labeled and fluorescently imaged. According to the DDW (Drug Discovery World) survey, only 7% of researchers are gradually shifting the two-dimensional cell culture method to three-dimensional culture. 7% of staff are expecting and positive, while 86% refuse to use it. The main reason is that they require The three-dimensional cell research method should be consistent with the traditional two-dimensional cell research method in the whole experimental system: no special tools and techniques are needed for the culture method, and the detection method can rely on the off-the-shelf instruments and methods for real-time detection. Detection.

建立三维细胞培养***需要考虑实际研究中的多重因素。如材料基质的来源（天然或合成材料）、材料基质的物化性能（化学相容性、机械性能、降解性、结构特性等）、材料基质的生物活性（粘附位点、诱导信号等），三维培养技术所需的设备、使用条件、适用范围，细胞的封装方式、培养方式、检测方式等等。理想的三维细胞培养***能够实现在细胞种植、培养、增殖、传代过程中以及后续的成像与定性，同传统两维细胞培养方式同样简便易行（easy as 2D) 。 Establishing a three-dimensional cell culture system requires consideration of multiple factors in actual research. Such as the source of the material matrix (natural or synthetic materials), the physical properties of the material matrix (chemical compatibility, mechanical properties, degradability, structural properties, etc.), the biological activity of the material matrix (adhesion sites, induction signals, etc.), The equipment, conditions of use, scope of application, cell packaging method, culture method, detection method, etc. required for three-dimensional culture technology. The ideal three-dimensional cell culture system enables imaging and characterization during cell planting, culture, proliferation, and passage, as well as traditional two-dimensional cell culture (easy as 2D).

再者，现代生物学、药学、医学的***研究需要涉及大量信息的获取和分析，为了降低研究成本，理想三维细胞培养***还能满足微尺度（microscale ) 、高通量 (high-throughput) 的研究要求。此外，为满足生物学、再生医学、组织工程、病理学等领域不同的研究目的，理想三维细胞培养***还应具备可图案化（patterning) 、微观结构（microstructure)可控性，易于监测（monitoring)等特点，实现具有更为复杂精细结构的仿生体外模型的建立。 Furthermore, systematic research in modern biology, pharmacy, and medicine involves the acquisition and analysis of a large amount of information. In order to reduce the cost of research, the ideal three-dimensional cell culture system can also meet microscale and high-throughput. Research requirements. In addition, in order to meet the different research purposes in the fields of biology, regenerative medicine, tissue engineering, pathology, etc., the ideal three-dimensional cell culture system should also have patterning, microstructure controllability, easy to monitor (monitoring) And so on, the establishment of a bionic in vitro model with more complex and fine structure.

随着生物医学、材料学、机械学、工程学等交叉学科的快速发展，越来越多的技术用于在空间上精确控制三维细胞培养微环境。例如诞生于半导体工业的微米尺度加工技术（如 3D打印机、激光雕刻机）被越来越广泛的应用于生物医学研究中以实现对于分子、材料和细胞在空间上的精确控制和高通量排列，其在建造图案化、高通量的三维微环境领域具有强大功能。利用此技术可重建体外仿生模型（如模拟血管的多层生理结构，以及肝小叶精细的生理结构），以及构建三维的分子、材料和细胞阵列芯片。再有，目前常用的高通量平台技术基于微孔板（如 96、 384孔板）或芯片（如基因、蛋白、材料和细胞芯片）形式，用自动化操作*** （如机械手、排枪、芯片点样***、个人点样仪等）执行实验过程，降低了所需试剂（如抗体、药物）、材料（如 matrigel、 collagen) 和细胞（如干细胞、肝细胞）的用量来实现微量样本研究。但是，这些技术的应用仅仅局限于具有拥有昂贵仪器并且有交叉学科（生物材料、工程制造、生物医学等）背景的实验室，其在传统生物学、药学和医学实验室的广泛应用尚存在技术上、资金上的瓶颈。因此能在常规生物学、医学或药学实验室实现的高通量图案化三维细胞微环境的建立将对于其广泛应用具有很重要的价值。 With the rapid development of interdisciplinary fields such as biomedicine, materials science, mechanics, and engineering, more and more technologies are being used to precisely control the three-dimensional cell culture microenvironment in space. For example, micron-scale processing technologies (such as 3D printers, laser engraving machines) born in the semiconductor industry are being widely used in biomedical research to achieve precise spatial control and high-throughput alignment of molecules, materials and cells. It has powerful capabilities in the construction of patterned, high-throughput 3D microenvironments. Using this technique, in vitro bionic models can be reconstructed (such as simulating the multi-layered physiological structure of blood vessels, as well as the fine physiological structure of the hepatic lobules), as well as constructing three-dimensional molecules, materials, and cell array chips. Furthermore, currently used high-throughput platform technologies are based on microplates (such as 96, 384-well plates) or chips (such as genes, proteins, materials, and cell chips), using automated operating systems (such as robots, rifles, and chip points). Sample system, personal spotter, etc.) Perform the experimental process, reducing the required reagents (such as antibodies, drugs), materials (such as matrigel, collagen) And the amount of cells (such as stem cells, liver cells) to achieve micro-sample studies. However, the application of these technologies is limited to laboratories with expensive instruments and interdisciplinary (biological materials, engineering, biomedical, etc.), and their technology is widely used in traditional biological, pharmaceutical and medical laboratories. The bottleneck in the capital. Therefore, the establishment of a high-throughput patterned three-dimensional cellular microenvironment that can be achieved in conventional biological, medical or pharmaceutical laboratories will be of great value for its widespread use.

综上所述三维细胞微环境构建的复杂性、目前已经商品化三维细胞培养产品的优缺点以及各领域研究需要，亟需一种能够和两维细胞培养方法一样简易，方便操作（easy as 2D) 的三维细胞培养方法，并同时满足图案化、高通量、实时无标记监测等研究目的。发明公开 In summary, the complexity of the three-dimensional cell microenvironment construction, the advantages and disadvantages of the commercially available three-dimensional cell culture products, and the research needs of various fields require a simple and convenient operation (easy as 2D). ) Three-dimensional cell culture methods, and at the same time meet the purpose of patterning, high-throughput, real-time label-free monitoring. Invention disclosure

本发明的目的是提供一种构建三维细胞微环境的方法及装置。 It is an object of the present invention to provide a method and apparatus for constructing a three-dimensional cellular microenvironment.

本发明所提供的构建三维微环境的装置是基于三维透明海绵支架的装置。所述海绵支架为透明的海绵支架，所述透明的海绵支架为透明度达到 50%以上的所述海绵支架。 The apparatus for constructing a three-dimensional microenvironment provided by the present invention is a device based on a three-dimensional transparent sponge stent. The sponge scaffold is a transparent sponge scaffold, and the transparent sponge scaffold is the sponge scaffold having a transparency of 50% or more.

所述海绵支架用生物材料制成，具有若干小孔；所述小孔的孔径为 lnm -999μηι, 孔间距为 1μηι-999μηι，所述小孔在所述海绵支架上所形成的孔隙率（多孔物体的孔隙体积和物体的总体积之比）为 70%-99.9%；所述海绵支架的体积为 0.^m³-1000cm³。 The sponge stent is made of a biological material and has a plurality of small holes; the pores have a pore diameter of 1 nm - 999 μηι, a pore spacing of 1 μηι - 999 μηι, and a porosity formed by the pores on the sponge stent (porous The ratio of the pore volume of the object to the total volume of the object is 70% - 99.9%; the volume of the sponge scaffold is 0. ^ m ^{3 -} 1000 cm ³ .

在本发明中，所述小孔的孔径具体可为 1μηι -150μηι、 1μηι -85μηι 10μηι -150μηι或 ΙΟμηι -125μηΐ; 所述孔隙率具体可为 82.4%-94.2%、 82.4%-93.3%或 93.3%-94.2% (如 82.4% 93.3%或 94.2% ) ；所述海绵支架的体积具体可为 0.2355mm³-37.56mm³、 0.2355mm³-4.82 mm³、 6.26mm³-37.56mm³、 0.603 mm³-4.82 mm³或 0.2355mm³-0.603 mm³ (具体如 0.2355mm³、 0.603 mm³、 1.204 mm³、 1.809 mm³、 2.415 mm³、 3.026 mm³、 3.620 mm³、 4.22 mm³、 4.82 mm³、 6.26mm³ 12.52 mm³、 25.04mm³或 37.56mm³) 。在本发明中，所述海绵支架的吸水量为理论体积的 1-15倍、 10-15倍、 3.5-5倍或 1-2倍。 In the present invention, the pore size of the pores may specifically be 1μηι -150μηι, 1μηι -85μηι 10μηι -150μηι or ΙΟμηι -125μηΐ; the porosity may specifically be 82.4%-94.2%, 82.4%-93.3% or 93.3% -94.2% (such as 82.4% 93.3% or 94.2%); the volume of the sponge holder may be 0.2355mm ³ -37.56mm ³ , 0.2355mm ³ -4.82 mm ³ , 6.26mm ³ -37.56mm ³ , 0.603 mm ³ -4.82 mm ³ or 0.2355 mm ³ -0.603 mm ³ (specifically eg 0.2355mm ³ , 0.603 mm ³ , 1.204 mm ³ , 1.809 mm ³ , 2.415 mm ³ , 3.026 mm ³ , 3.620 mm ³ , 4.22 mm ³ , 4.82 mm ³ , 6.26mm ³ 12.52 mm ³ , 25.04mm ³ or 37.56mm ³ ). In the present invention, the sponge scaffold has a water absorption amount of 1-15 times, 10-15 times, 3.5-5 times or 1-2 times of the theoretical volume.

所述生物材料为可交联的人工合成的生物材料和 /或可交联的天然生物材料;所述人工合成的生物材料为下述至少一种：聚乙二醇、聚乙二醇衍生物、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、聚縮醛、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、聚碳酸酯和聚氧化乙烯；所述天然生物材料为下述至少一种：明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、蛋白多糖、糖蛋白、透明质酸、层连接蛋白和纤维连接蛋白。 The biomaterial is a crosslinkable synthetic biomaterial and/or a crosslinkable natural biomaterial; the synthetic biomaterial is at least one of the following: polyethylene glycol, polyethylene glycol derivative , 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, and polyethylene oxide; the natural biomaterial is at least one of the following: gelatin, Gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycans, glycoproteins, hyaluronic acid, laminin and fibronectin.

本发明所提供的构建三维微环境的装置，包括下述 A1 ) 和 A2) 的两个器件： A1 ) 所述透明的海绵支架或由两个以上所述透明的海绵支架组成的海绵支架阵列； A2)用于装载样品液的基底 A; The apparatus for constructing a three-dimensional microenvironment provided by the present invention comprises two devices of the following A1) and A2): A1) the transparent sponge scaffold or an array of sponge scaffolds composed of two or more transparent sponge scaffolds; A2) a substrate A for loading a sample liquid;

所述装载样品液的基底 A为亲水性基底或疏水性基底；所述亲水性基底或疏水性基底由各种改性方法制备，包括化学修饰法、物理改性法以及将其结合使用。所述基底的亲水性或疏水性使样品液在所述基底的表面形成液体薄层；所述液体薄层的厚度通常为 1μηι -200μηι, 既可形成细胞薄层，又不会在重力作用下形成聚集，导致分布不均。所述亲水性或疏水性基底的微加工技术为下述至少一种：影印石版术（photolithography) 、微接触打印技术（microcontact printing) 、微流体图案技术 (microfluidic patterning) 层流图案技术 (laminar flow patterning) 模版图案技术 (stencil patterning) 压印光刻技术 (Imprint lithography)、流体光刻技术 (flow lithography)等。在本发明中，所述液体薄层的厚度具体可为 ΙΟμηι -ΙΟΟμηι或 10μηι-50μηι，如 ΙΟμηι或 50μηι。 The substrate A carrying the sample liquid is a hydrophilic substrate or a hydrophobic substrate; the hydrophilic substrate or the hydrophobic substrate is prepared by various modification methods, including chemical modification, physical modification, and combination thereof. . The hydrophilicity or hydrophobicity of the substrate causes the sample liquid to form a thin layer of liquid on the surface of the substrate; the thickness of the liquid layer is usually 1 μηι - 200 μηι, which can form a thin layer of cells without gravity Aggregation occurs underneath, resulting in uneven distribution. The micromachining technique of the hydrophilic or hydrophobic substrate is at least one of the following: photolithography, microcontact printing, microfluidic patterning layer Laminar flow patterning stencil patterning Imprint lithography, flow lithography, etc. In the present invention, the thickness of the liquid thin layer may specifically be ΙΟμηι -ΙΟΟμηι or 10μηι-50μηι, such as ΙΟμηι or 50μηι.

所述生物材料的交联方法为下述至少一种：光交联法、化学交联法、物理交联法、辐射交联法、酶催化交联法、活化微珠交联法等。 The crosslinking method of the biomaterial is at least one of the following: a photocrosslinking method, a chemical cross-linking method, a physical cross-linking method, a radiation cross-linking method, an enzyme-catalyzed cross-linking method, an activated microbead cross-linking method, and the like.

所述小孔的制孔技术为下述至少一种：致孔剂（porogen)滤除法、相分离法、乳液冷冻干燥法、溶剂蒸发法、气体泡沫法、纤维粘合法等。 The pore-forming technique of the pores is at least one of the following: porogen filtration, phase separation, emulsion freeze-drying, solvent evaporation, gas foaming, fiber bonding, and the like.

在制备所述透明的海绵支架的过程中，需要使用透明剂，改变所述生物材料的光学性能，以达到透明的效果。同时还可以通过使用微加工技术，实现所述海绵支架的图案化，和 /或实现所述海绵支架阵列的高通量。所述三维微环境的微加工技术为下述至少一禾中：影印石版术 (photolithography) 、微接触打印技术 ( microcontact printing) 、微流体图案技术 (microfluidic patterning)、层流图案技术 (laminar flow patterning)、模板图案技术 (stencil patterning) 压印光亥 [J技术 (Imprint lithography) 流体光亥 [J技术 (flow lithography) 等。在本发明中，具体通过使用光掩膜，根据需要设计透光部分的图案，进而通过光交联得到图案化的三维海棉支架以及高通量的三维海绵支架阵列。 In the process of preparing the transparent sponge scaffold, it is necessary to use a transparent agent to change the optical properties of the biomaterial to achieve a transparent effect. At the same time, the patterning of the sponge scaffold can be achieved by using micromachining techniques, and/or high throughput of the sponge scaffold array can be achieved. The micromachining technology of the three-dimensional microenvironment is at least one of the following: photolithography, microcontact printing, microfluidic patterning, laminar flow patterning. ), stencil patterning, imprint lithography, fluid lithography, etc. In the present invention, by designing a pattern of a light-transmitting portion as needed by using a photomask, a patterned three-dimensional sponge holder and a high-throughput three-dimensional sponge stent array are obtained by photocrosslinking.

所述海绵支架阵列由三个以上的所述海绵支架组成，形成所述高通量的三维海绵支架阵列。在本发明中，组成所述海绵支架阵列的所述海绵支架具体可为 16-192个，如 16、 24、 64、 192个。 The sponge scaffold array is composed of more than three of the sponge scaffolds to form the high throughput three dimensional sponge scaffold array. In the present invention, the sponge scaffolds constituting the sponge scaffold array may specifically be 16-192, such as 16, 24, 64, 192.

本发明中，所述透明的海绵支架的制备有光交联法和化学交联法。 In the present invention, the transparent sponge scaffold is prepared by a photocrosslinking method and a chemical cross-linking method.

在本发明的一个实施例中，采用光交联法制备所述透明的海绵支架，包括如下步骤: bl )将聚合物单体聚乙二醇二丙烯酸酯和光引发剂 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮溶解在所述透明剂与水的混合溶液中，得到可光交联的预聚物溶液 A; In one embodiment of the invention, the transparent sponge scaffold is prepared by photocrosslinking, comprising the steps of: bl) polymer monomer polyethylene glycol diacrylate and photoinitiator 2-hydroxy-4-( Dissolving 2-hydroxyethoxy)-2-methylpropiophenone in a mixed solution of the clearing agent and water to obtain a photocrosslinkable prepolymer solution A;

b2)利用紫外光源照射步骤 bl )获得的可光交联的预聚物溶液 A，使所述可光交联的预聚物溶液 A发生交联反应，得到水凝胶； B2) irradiating the photocrosslinkable prepolymer solution A obtained in step bl) with an ultraviolet light source to cause a cross-linking reaction of the photocrosslinkable prepolymer solution A to obtain a hydrogel;

b3 )将步骤 b2)获得的所述水凝胶浸于超纯水中除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质。 B3) The hydrogel obtained in the step b2) is immersed in ultrapure water to remove the uncrosslinked polymer monomer polyethylene glycol diacrylate, the clearing agent and impurities.

在上述方法中，所述聚合物单体聚乙二醇二丙烯酸酯在所述可光交联的预聚物溶液 A中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 l-50g所述聚合物单体聚乙二醇二丙烯酸酯；所述光引发剂 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮在所述可光交联的预聚物溶液 A中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 0.1-10g所述 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮；所述透明剂在所述透明剂与水的混合溶液中的体积百分含量需大于等于 0.01%，同时小于 100%。 In the above method, the content of the polymer monomer polyethylene glycol diacrylate in the photocrosslinkable prepolymer solution A is per 100 ml of the photocrosslinkable prepolymer solution A. Containing 1-50 g of the polymer monomer polyethylene glycol diacrylate; the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the photocrossing The content of the prepolymer solution A is 0.1-10 g of the 2-hydroxy-4-(2-hydroxyethoxy)-2-methyl group per 100 ml of the photocrosslinkable prepolymer solution A. The phenylacetone; the volume fraction of the transparent agent in the mixed solution of the transparent agent and water needs to be 0.01% or more and less than 100%.

所述透明剂可为 1,2,4-丁三醇、乙二醇， 1,3-丁二醇，丙三醇， 1,2-丙二醇， 1,3-丙二醇，季戊四醇，顺 -1,2-环戊二醇，丁四醇和戊五醇等多元醇中的至少一种。 The transparent agent may be 1,2,4-butanetriol, ethylene glycol, 1,3-butanediol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol, cis-1, At least one of a polyhydric alcohol such as 2-cyclopentanediol, tetramethylene alcohol or pentaerythritol.

在本发明的一个实施例中，所述聚合物单体聚乙二醇二丙烯酸酯在所述可光交联的预聚物溶液 A中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 10g所述聚合物单体聚乙二醇二丙烯酸酯；所述光引发剂 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮在所述可光交联的预聚物溶液 A中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 0.5g 所述 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮；所述透明剂具体为 1,2,4-丁三醇，所述 1,2,4- 丁三醇在所述透明剂与水的混合溶液中的体积百分含量具体为 60% (即所述 1,2,4-丁三醇与所述水的体积比为 3: 2) 。 In one embodiment of the present invention, the polymer monomer polyethylene glycol diacrylate is contained in the photocrosslinkable prepolymer solution A in an amount of the photocrosslinkable prepolymerized per 100 ml. Solution A contains 10 g of the polymer monomer polyethylene glycol diacrylate; the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone is The content of the photocrosslinked prepolymer solution A is 0.5 g per 100 ml of the photocrosslinkable prepolymer solution A. The 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone; the transparent agent is specifically 1,2,4-butanetriol, the 1,2,4-butane The volume percentage of the alcohol in the mixed solution of the clearing agent and water is specifically 60% (i.e., the volume ratio of the 1,2,4-butanetriol to the water is 3:2).

在本发明的另一个实施例中，采用化学交联法制备所述透明的海绵支架，包括如下步骤： In another embodiment of the invention, the transparent sponge scaffold is prepared by chemical cross-linking, comprising the steps of:

cl )将聚合物单体聚乙二醇二丙烯酸酯溶解在所述透明剂与水的混合溶液中，再加入过硫酸铵和 Ν,Ν,Ν',Ν'-四甲基二乙胺，得到预聚物溶液 Β; Cl) dissolving the polymer monomer polyethylene glycol diacrylate in a mixed solution of the transparent agent and water, and then adding ammonium persulfate and hydrazine, hydrazine, hydrazine, Ν'-tetramethyldiethylamine, Obtaining a prepolymer solution Β;

c2) 在步骤 cl ) 得到预聚物溶液 B后，将其加到制备海绵支架的模具中，使所述预聚物溶液 B发生化学交联反应（室温下进行），得到水凝胶； C2) after obtaining the prepolymer solution B in step cl), adding it to a mold for preparing a sponge scaffold, and subjecting the prepolymer solution B to a chemical cross-linking reaction (at room temperature) to obtain a hydrogel;

c3)将步骤 c2)获得的所述水凝胶浸于超纯水中除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质。 C3) The hydrogel obtained in the step c2) is immersed in ultrapure water to remove the uncrosslinked polymer monomer polyethylene glycol diacrylate, the clearing agent and impurities.

在上述方法中，所述聚合物单体聚乙二醇二丙烯酸酯在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 l-50g所述聚合物单体聚乙二醇二丙烯酸酯；所述过硫酸铵在所述预聚物溶液 B 中的含量可为每 100ml 所述预聚物溶液 B 中含有 0.01-lg所述过硫酸铵；所述 Ν,Ν,Ν',Ν'-四甲基二乙胺在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 0.01-lg所述 Ν,Ν,Ν',Ν'-四甲基二乙胺；所述透明剂在所述透明剂与水的混合溶液中的体积百分含量需大于等于 0.01%，同时小于 100%。 In the above method, the polymer monomer polyethylene glycol diacrylate may be contained in the prepolymer solution B in an amount of 1 to 50 g of the polymer monomer per 100 ml of the prepolymer solution B. a polyethylene glycol diacrylate; the ammonium persulfate content in the prepolymer solution B may be 0.01-lg of the ammonium persulfate per 100 ml of the prepolymer solution B; , Ν,Ν', Ν'-tetramethyldiethylamine may be contained in the prepolymer solution B in an amount of 0.01-lg per 100 ml of the prepolymer solution B. And Ν'-tetramethyldiethylamine; the volume percentage of the clearing agent in the mixed solution of the transparent agent and water needs to be 0.01% or more and less than 100%.

在本发明的一个实施例中，所述聚合物单体聚乙二醇二丙烯酸酯在所述预聚物溶液 In one embodiment of the invention, the polymer monomer polyethylene glycol diacrylate is in the prepolymer solution

Β中的含量可为每 100ml所述预聚物溶液 Β中含有 10g所述聚合物单体聚乙二醇二丙烯酸酯；所述过硫酸铵在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 0.05g所述过硫酸铵；所述 Ν,Ν,Ν',Ν'-四甲基二乙胺在所述预聚物溶液 Β中的含量可为每 100ml所述预聚物溶液 B中含有 0.5g所述 Ν,Ν,Ν',Ν'-四甲基二乙胺。所述透明剂具体为 1,2,4-丁三醇，所述 1,2,4-丁三醇在所述透明剂与水的混合溶液中的体积百分含量为 60% (即所述 1,2,4-丁三醇与所述水的体积比为 3: 2) 。 The content of the cerium may be 10 g of the polymer monomer polyethylene glycol diacrylate per 100 ml of the prepolymer solution; the content of the ammonium persulfate in the prepolymer solution B may be Each 100 ml of the prepolymer solution B contains 0.05 g of the ammonium persulfate; the content of the ruthenium, osmium, iridium, Ν'-tetramethyldiethylamine in the ruthenium of the prepolymer solution may be Each 100 ml of the prepolymer solution B contained 0.5 g of the hydrazine, hydrazine, hydrazine, Ν'-tetramethyldiethylamine. The transparent agent is specifically 1,2,4-butanetriol, and the volume percentage of the 1,2,4-butanetriol in the mixed solution of the transparent agent and water is 60% (ie, the The volume ratio of 1,2,4-butanetriol to the water is 3: 2).

在上述化学交联方法中，所述制备海绵支架的模具具体可用生物材料制成，所述生物材料为人工合成的生物材料和 /或天然生物材料;所述人工合成的生物材料为下述至少一种：聚甲基丙烯酸酯、聚乙二醇、聚乙二醇衍生物、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、聚縮醛、聚氰基丙烯酸酯、聚氨基甲酸酯、聚吡咯、聚酯、聚乙烯、聚碳酸酯和聚氧化乙烯；所述天然生物材料为下述至少一种：明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、蛋白多糖、糖蛋白、透明质酸、层连接蛋白和纤维连接蛋白。 In the above chemical crosslinking method, the mold for preparing the sponge stent may be specifically made of a biological material, which is a synthetic biomaterial and/or a natural biomaterial; the artificial biomaterial is at least One: polymethacrylate, polyethylene glycol, polyethylene glycol derivative, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid copolymer, polydimethyl Silicone, polyanhydride, polyacrylate, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polyethylene, polycarbonate, and polyethylene oxide; The natural biomaterial is at least one of the following: gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, laminin and fiber Connexin.

在上述光交联法和化学交联法制备所述透明的海绵支架的步骤中，在步骤 b3 ) 和 c3) 中所述除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质后，还包括将所述水凝胶在 -200°C~0°C条件下冷冻 l-72h，再干燥 l-72h，得到所述海绵支架或所述海绵支架阵列。在本发明的实施例中，在除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质后，将所述水凝胶在 -20°C条件下冷冻 4-5h，再在 -50°C， 20pa条件下，干燥 12小时，得到所述海绵支架或所述海绵支架阵列。 In the step of preparing the transparent sponge scaffold by the above photocrosslinking method and chemical cross-linking method, the uncrosslinked polymer monomer polyethylene glycol diacrylate is removed as described in steps b3) and c3) After the transparent agent and the impurities, the hydrogel is further frozen at -200 ° C ~ 0 ° C for 1-72 h, and then dried for l-72 h to obtain the sponge scaffold or the sponge scaffold array. . In an embodiment of the invention, after removing the uncrosslinked polymer monomer polyethylene glycol diacrylate, the clearing agent and impurities, the hydrogel is frozen at -20 ° C. 4-5h, and then dried at -50 ° C, 20 Pa, for 12 hours, to obtain the sponge stent or the sponge stent array.

根据实际需要，本发明所提供的用于构建三维微环境的装置，还包括镶嵌在所述基底 A上的边框。 According to actual needs, the apparatus for constructing a three-dimensional microenvironment provided by the present invention further includes a bezel mounted on the substrate A.

所述边框用生物材料制成，所述生物材料为人工合成的生物材料和 /或天然生物材料；所述人工合成的生物材料为下述至少一种：聚乙二醇、聚乙二醇衍生物、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、聚縮醛、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、聚碳酸酯和聚氧化乙烯；所述天然生物材料为下述至少一种：明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、蛋白多糖、糖蛋白、透明质酸、层连接蛋白和纤维连接蛋白。 The frame is made of a biological material, which is a synthetic biomaterial and/or a natural biomaterial; the synthetic biomaterial is at least one of the following: polyethylene glycol, polyethylene glycol derived , polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polycondensation An aldehyde, a polycyanoacrylate, a polyurethane, a polypyrrole, a polyester, a polymethacrylate, a polyethylene, a polycarbonate, and a polyethylene oxide; the natural biomaterial is at least one of the following: gelatin , gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, proteoglycans, glycoproteins, hyaluronic acid, laminin and fibronectin.

在本发明的一个实施例中，所述基底 A具体为亲水性基底；所述基底 A上的边框具体为用聚甲基丙烯酸甲酯制成的。 In one embodiment of the invention, the substrate A is specifically a hydrophilic substrate; the frame on the substrate A is specifically made of polymethyl methacrylate.

实际操作中，根据需要，所述装置还包括支撑所述海绵支架或所述海绵支架阵列的基底 B。在本发明的一个实施例中，所述基底 B为固定所述海绵支架或所述海绵支架阵列的载玻片。 In actual operation, the device further includes a substrate B supporting the sponge holder or the sponge holder array as needed. In one embodiment of the invention, the substrate B is a slide that holds the sponge holder or the sponge holder array.

所述海绵支架或所述装置在构建三维微环境中的应用也属于本发明的保护范围。本发明所提供的构建三维微环境的方法是基于所述海绵支架（三维透明海绵支架），采用与传统两维细胞培养方法一样简便易行的方法，可实现包含可溶性因子、生物材料和细胞的三维细胞微环境的构建，并同时满足图案化、高通量、无标记实时监测等研究目的。（图 1 ) The use of the sponge scaffold or the device in constructing a three-dimensional microenvironment is also within the scope of the present invention. The method for constructing a three-dimensional microenvironment provided by the present invention is based on the sponge scaffold (three-dimensional transparent sponge scaffold), and is capable of realizing a method comprising a soluble factor, a biological material and a cell, as well as a conventional two-dimensional cell culture method. The construction of three-dimensional cell microenvironment, and at the same time meet the research purposes of patterning, high-throughput, label-free real-time monitoring. (figure 1 )

本发明所述的与传统两维细胞培养方法一样简单易行的种植方法基于传统滴加法和液体薄层法。传统滴加方法是指将样品液体直接滴加在支架表面或旁侧，样品液自动被吸入支架内部。液体薄层法是指将三维海绵支架接触覆盖在分子、材料和细胞的液体薄层上，利用海绵支架的自动吸附作用，使得分子、材料和细胞自动分散进入到所述海绵支架中，以实现高通量同步装载，完成分子、材料和细胞三维微环境的构建。 The planting method of the present invention which is as simple as the conventional two-dimensional cell culture method is based on the conventional dropping method and the liquid thin layer method. The conventional dropping method means that the sample liquid is directly dropped on the surface or the side of the stent, and the sample liquid is automatically sucked into the inside of the stent. The liquid thin layer method refers to contacting a three-dimensional sponge scaffold on a thin layer of liquid covering molecules, materials and cells, and using the automatic adsorption of the sponge scaffold, the molecules, materials and cells are automatically dispersed into the sponge scaffold to realize High-throughput simultaneous loading completes the construction of molecules, materials and cellular three-dimensional microenvironments.

所述构建三维微环境的液体薄层法具体为用所述装置构建三维微环境，可包括如下步骤： The liquid thin layer method for constructing a three-dimensional microenvironment is specifically to construct a three-dimensional microenvironment by using the device, which may include the following steps:

al ) 将样品液置于所述装置的基底 A上，形成液体薄层； Al) placing a sample liquid on the substrate A of the device to form a thin layer of liquid;

a2) 将步骤 al ) 中形成所述液体薄层的所述基底 A覆盖在所述装置的海绵支架或海绵支架阵列上，或者将所述海绵支架或所述海绵支架阵列覆盖在步骤 al )中形成所述液体薄层的所述基底 A上，待所述样品液分散进入到所述海绵支架或所述海绵支架阵列后，实现所述样品液的三维尺度装载，完成所述三维微环境的构建； A2) covering the substrate A forming the thin layer of the liquid in step a1 on the sponge or sponge scaffold array of the device, or covering the sponge scaffold or the sponge scaffold array in step a) Forming the thin layer of the liquid on the substrate A, after the sample liquid is dispersed into the sponge scaffold or the sponge scaffold array, achieving three-dimensional loading of the sample liquid to complete the three-dimensional microenvironment Construct;

所述液体薄层的厚度为 Ιμηι -200μηι, 既可形成细胞薄层，又不会在重力作用下形成聚集，导致分布不均。在本发明中，所述液体薄层的厚度具体可为 ΙΟμηι -ΙΟΟμηι或 10μιη-50μιη, 如 ΙΟμιη或 50μιη。 The thin layer of liquid has a thickness of Ιμηι - 200μηι, which can form a thin layer of cells without forming agglomeration under the action of gravity, resulting in uneven distribution. In the present invention, the thickness of the liquid thin layer may specifically be ΙΟμηι - ΙΟΟμηι or 10μηη - 50μηη, such as ΙΟμηη or 50μιη.

在上述方法中，所述样品液具体可包括下述 a) -d) 中任一所述的物质： a)各种分子物质（如小分子化合物、药物、核酸、蛋白等）中的任一种或任几种的混合物； b ) 各种天然与合成材料（如细胞外基质，高分子材料、微珠等）中的任一种或任几种的混合物； c )各种细胞和微生物（如真核 /原核细胞、病毒、微生物等）中的任一种或任几种的混合物； d) a) -c ) 中任几种的混合物。 In the above method, the sample liquid may specifically include any one of the following a) - d): a) various points Any one or a mixture of any of the sub-substances (eg, small molecule compounds, drugs, nucleic acids, proteins, etc.); b) various natural and synthetic materials (eg, extracellular matrices, polymeric materials, microbeads, etc.) Any one or a mixture of any of the following; c) any one or a mixture of any of a variety of cells and microorganisms (eg, eukaryotic/prokaryotic cells, viruses, microorganisms, etc.); d) a) -c) a mixture of several of them.

在本发明中，上述所有三维微环境均为包括下述 a) -d) 中任一所述的三维微环境： a) 各种分子物质（如小分子化合物、药物、核酸、蛋白等）中的任一种或任几种的混合物； b ) 各种天然与合成材料（如细胞外基质，高分子材料、微珠等）中的任一种或任几种的混合物； c )各种细胞和微生物（如真核 /原核细胞、病毒、微生物等）中的任一种或任几种的混合物； d) a) -c ) 中任几种的混合物。 In the present invention, all of the above three-dimensional microenvironments include the three-dimensional microenvironment described in any one of the following a) - d): a) various molecular substances (such as small molecule compounds, drugs, nucleic acids, proteins, etc.) Any one or a mixture of any of the following; b) any one or a mixture of any of a variety of natural and synthetic materials (eg, extracellular matrices, polymeric materials, microbeads, etc.); c) various cells And a mixture of any one or any of microorganisms (such as eukaryotic/prokaryotic cells, viruses, microorganisms, etc.); d) a mixture of any of a)-c).

上述三维细胞微环境研究应用领域广泛，包括但不限于：分子 /材料 /细胞的芯片用于研究分子 /细胞、材料 /细胞、细胞 /细胞相互作用；药物筛选；体外模型构建；组织工程；再生医学；病理研究等等。 The above three-dimensional cellular microenvironment research applications are extensive, including but not limited to: molecular/material/cell microarrays for studying molecules/cells, materials/cells, cell/cell interactions; drug screening; in vitro model building; tissue engineering; regeneration Medicine; pathology research and so on.

附图说明 DRAWINGS

图 1为方法设计总图。其中， A-1至 A-3为两维细胞培养技术，具体的， A-1为细胞滴加平铺在培养皿中， A-2为显微镜观察培养皿上细胞状态， A-3为传代培养，继 A-3 之后，可根据需要进行各种细胞研究。 B-1 至 B-5 为三维细胞培养技术，具体的， B-1 为细胞自动吸附进入透明多孔海绵支架， B-2为显微镜观察透明海绵支架内部细胞状态， B-3为传代培养， B-4为液体薄层法高通量自动装载（1-液体薄层； 2-海绵支架阵列； 3- 疏水性边框）， B-5为微加工技术实现微尺度、图案化设计（1-TMP修饰玻片； 2-盖玻片； 3-OTS修饰玻片； 4-紫外光； 5-图案化光掩膜； 6-预聚物），继 B-5之后，可根据需要进行各种细胞研究。 Figure 1 is a general view of the method design. Among them, A-1 to A-3 are two-dimensional cell culture techniques. Specifically, A-1 is a cell dropping and tiling in a culture dish, A-2 is a microscopic observation of the cell state on the culture dish, and A-3 is subculture. After A-3, various cell studies can be performed as needed. B-1 to B-5 are three-dimensional cell culture techniques. Specifically, B-1 is the cell that is automatically adsorbed into the transparent porous sponge scaffold, B-2 is the microscopic observation of the internal cell state of the transparent sponge scaffold, and B-3 is subcultured, B -4 is a high-throughput automatic loading of liquid thin layer method (1-liquid thin layer; 2-sponge holder array; 3- hydrophobic frame), B-5 is micro-machining technology for micro-scale, patterned design (1-TMP Modified slides; 2-cover slides; 3-OTS modified slides; 4-UV light; 5-patterned photomask; 6-prepolymer), after B-5, various cells can be used as needed the study.

图 2为透明海绵支架制备及表征。 A为化学交联法制备海绵支架模具。 B为化学交联法制备海绵支架实物图（正面图和侧面图）及其电镜图。 C和 D为光交联法制备海绵支架实物图（圆柱形和圆环形）及其电镜图（C大孔径， D小孔径）。 E和 F为光交联法制备海绵支架孔径分布图（E大孔径， F小孔径）。 Figure 2 shows the preparation and characterization of a transparent sponge scaffold. A is a sponge stent mold prepared by chemical crosslinking method. B is a physical diagram of the sponge scaffold prepared by the chemical cross-linking method (front view and side view) and its electron micrograph. C and D are photo-crosslinking methods for the preparation of sponge scaffolds (cylindrical and toroidal) and their electron micrographs (C large aperture, D small aperture). E and F are the pore size distribution maps of the sponge scaffold prepared by photocrosslinking (E large pore size, F small pore diameter).

图 3 为透明海绵支架自动装载分子、材料、细胞。 A和 B为圆柱形海绵支架阵列自动装载分子（酚红溶液）， A为装载前， B为装载后。 C和 D为正六边形海绵支架阵列自动装载材料（明胶）， C为装载前， D为装载后。 E为正六边形和长方形海绵支架阵列自动装载细胞（HeLa) 。 F-1至 F-9为海绵支架自动装载荧光微球动态图（荧光微球直径 ΙΟμηι) 。 Figure 3 shows a transparent sponge scaffold that automatically loads molecules, materials, and cells. A and B are cylindrical sponge scaffold arrays that automatically load molecules (phenol red solution), A for loading and B for loading. C and D are regular hexagonal sponge bracket arrays for automatic loading of materials (gelatin), C for loading and D for loading. E is a regular hexagonal and rectangular sponge scaffold array of autoloaded cells (HeLa). F-1 to F-9 are dynamic maps of the fluorescent microspheres (fluorescent microsphere diameter ΙΟμηι) automatically loaded on the sponge scaffold.

图 4 为透明海绵支架光学性能测试。 Α 为透过化学交联法制备的透明海绵支架 ( 0.5mm)观察文字（左侧两个样品为 0%1,2,4-丁三醇，右侧两个样品为 60%1,2,4-丁三醇）。 B为化学交联法制备不同高度的透明海绵支架。 C为透过光交联法制备海绵支架观察文字（0%1,2,4-丁三醇）。 D为透过光交联法制备透明海绵支架观察文字（60%1,2,4- 丁三醇）。 E 为不同体积比的透明剂 1,2,4-丁三醇对透明海绵支架透明度的定量图。 F 为不同高度对透明海绵支架（60%透明剂 1,2,4-丁三醇）透明度的定量图。 Figure 4 shows the optical performance test of the transparent sponge holder.观察 Observe the text for the transparent sponge scaffold (0.5mm) prepared by chemical cross-linking method (the two samples on the left are 0% 1,2,4-butanetriol, and the two samples on the right are 60% 1,2, 4-butanetriol). B is a transparent sponge scaffold of different heights prepared by chemical cross-linking method. C is a sponge scaffold prepared by photocrosslinking. The text (0% 1,2,4-butanetriol) is observed. D is a transparent sponge scaffold prepared by photocrosslinking to observe the text (60% 1,2,4-butanetriol). E is a quantitative diagram of the transparency of the transparent sponge scaffold with different volume ratios of 1,2,4-butanetriol. F is a quantitative plot of the transparency of a transparent sponge scaffold (60% clear 1,2,4-butanetriol) at different heights.

图 5为 HeLa细胞在透明海绵支架中生长形成肿瘤微球。 A-1至 A-5: 显微镜观察 HeLa细胞随时间在透明海绵支架中生长形成肿瘤微球。 B为显微镜观察 HeLa肿瘤微球的荧光图片。 C为 HeLa肿瘤微球的电镜图。 D为激光共聚焦显微镜观察 HeLa肿瘤微球的三维荧光图片。 Figure 5 shows the growth of HeLa cells in a transparent sponge scaffold to form tumor microspheres. A-1 to A-5: Microscopic observation of HeLa cells grows in a transparent sponge scaffold over time to form tumor microspheres. B is a microscope to observe HeLa tumor microspheres Fluorescent picture. C is an electron micrograph of HeLa tumor microspheres. D is a three-dimensional fluorescence picture of HeLa tumor microspheres observed by laser confocal microscopy.

图 6为 HeLa肿瘤微球传代培养保持高存活率。 A为 HeLa肿瘤微球在透明支架中的显微镜图片。 B为 HeLa肿瘤微球被消化洗涤出支架。 C和 D为肿瘤微球死 /活染色荧光图片， C为低倍镜 2x， D为高倍镜 10x。 Figure 6 shows the high survival rate of HeLa tumor microspheres subculture. A is a microscopic picture of HeLa tumor microspheres in a transparent scaffold. B is a HeLa tumor microsphere that is digested and washed out of the scaffold. C and D are tumor microspheres dead/live staining fluorescence pictures, C is low magnification 2x, and D is high magnification 10x.

图 7为液体薄层法自动装载分子、材料进入高通量、图案化海绵支架。 A-1和 A-2 为等离子清洗法形成亲水性基底， A-1为清洗前水的接触角， A-2为清洗后水的接触角。 B为微加工技术（影印石版术）制备高通量海绵支架阵列。 C为等离子清洗法和微接触打印法形成不同染料液体薄层。 D为液体薄层法高通量自动装载不同染料液体进入海绵支架阵列。 E为液体薄层法自动装载 Doxorubicin药物和荧光微球进入微加工技术（影印石版术）制备的图案化海绵支架（清华大学百年校庆图标）。 Figure 7 shows a liquid thin layer method for automatically loading molecules and materials into a high-throughput, patterned sponge scaffold. A-1 and A-2 form a hydrophilic substrate for plasma cleaning, A-1 is the contact angle of water before washing, and A-2 is the contact angle of water after washing. B is a micromachining technology (photolithography) to prepare a high-throughput sponge stent array. C is a thin layer of different dye liquids for plasma cleaning and microcontact printing. D is a liquid thin layer method for high throughput automatic loading of different dye liquids into the sponge scaffold array. E is a liquid thin layer method for automatically loading Doxorubicin drugs and fluorescent microspheres into micromachining technology (photolithography) to prepare a patterned sponge scaffold (Tsinghua University Centennial Celebration icon).

图 8为液体薄层法自动装载 HeLa细胞进入海绵支架阵列。 A为液体薄层法自动装载 HeLa细胞进入圆环形海绵支架阵列。 B-1至 B-3为不同细胞浓度液体薄层自动装载效果图。 C为不同细胞浓度液体薄层自动装载进入海绵支架内部的激光共聚焦显微镜三维扫描图。 D为 Cell Titer-Blue测试液体薄层法自动装载不同浓度 HeLa细胞进入海绵支架的细胞量与液体薄层残余细胞量。 E为 Cell Titer-Blue定量荧光强度-活细胞数量标准曲线。 F为根据标准曲线 E图，定量液体薄层法自动装载不同浓度 HeLa细胞进入海绵支架的比率。 Figure 8 is a liquid thin layer method for automatically loading HeLa cells into a sponge scaffold array. A is a liquid thin layer method to automatically load HeLa cells into an array of circular sponge stents. B-1 to B-3 are automatic loading effects of liquid layers at different cell concentrations. C is a three-dimensional scan of a laser confocal microscope that automatically loads a thin layer of liquid at different cell concentrations into the interior of the sponge scaffold. D is the cell Titer-Blue test liquid thin layer method to automatically load different concentrations of HeLa cells into the sponge scaffold and the residual amount of liquid in the thin layer of liquid. E is the Cell Titer-Blue quantitative fluorescence intensity-live cell number standard curve. F is the ratio of the concentration of HeLa cells entering the sponge scaffold by the quantitative liquid thin layer method according to the standard curve E map.

实施发明的最佳方式 The best way to implement the invention

下述实施例中所使用的实验方法如无特殊说明，均为常规方法。 The experimental methods used in the following examples are all conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。 The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

下述实施例中所涉及的孔隙率均指多孔物体的孔隙体积和物体的总体积之比。实施例 1、分别采用化学交联法和光交联法制备图案化透明三维海绵支架本实施例所提供的制备透明海绵支架的生物材料为聚乙二醇二丙烯酸酯 (PEGDA4000) 。聚乙二醇二丙烯酸酯具有良好的生物相容性、机械性能、不可降解性以及适用多种交联方法等优点，适合作为本实施例制备透明海绵支架的生物材料。聚乙二醇二丙烯酸酯合成方法：在氮气条件下，将 5g聚乙二醇 (PEG4000)粉末搅拌溶解于 50ml二氯甲烷溶液中，缓慢逐滴加入 0.76ml的三乙胺溶液和 0.47ml的丙烯酰氯溶液，室温下搅拌反应 24h。之后，加入 100ml 2M的碳酸钾溶液洗涤，并静置分层，收集下层二氯甲烷混合液。将二氯甲烷混合液滴加入 500ml无水***溶剂中沉淀，过滤收集白色粉末，并室温干燥，最终得到白色 PEGDA固体粉末。 The porosity referred to in the following examples refers to the ratio of the pore volume of the porous body to the total volume of the object. Example 1. Preparation of a patterned transparent three-dimensional sponge scaffold by chemical cross-linking method and photocrosslinking method respectively The biomaterial for preparing a transparent sponge scaffold provided in this embodiment is polyethylene glycol diacrylate (PEGDA4000). Polyethylene glycol diacrylate has the advantages of good biocompatibility, mechanical properties, non-degradability and various crosslinking methods, and is suitable as a biological material for preparing a transparent sponge stent in this embodiment. Polyethylene glycol diacrylate synthesis method: Under nitrogen, 5g of polyethylene glycol (PEG4000) powder is stirred and dissolved in 50ml of dichloromethane solution, slowly adding 0.76ml of triethylamine solution and 0.47ml The acryloyl chloride solution was stirred at room temperature for 24 hours. Thereafter, it was washed by adding 100 ml of a 2 M potassium carbonate solution, and allowed to stand for separation, and the lower dichloromethane mixture was collected. The methylene chloride mixed droplets were precipitated by adding to 500 ml of anhydrous ether solvent, and the white powder was collected by filtration and dried at room temperature to give a white PEGDA solid powder.

(一）化学交联法制备透明海绵支架 (1) Preparation of transparent sponge scaffold by chemical cross-linking method

1. 激光切割法制备支架模具 1. Laser cutting method to prepare bracket mold

采用 Rayjet激光雕刻机切割厚度分别为 0.5mm、 lmm、 2mm、 3mm的聚甲基丙烯酸甲酯（PMMA) 平板形成模具。模板设计由软件 AutoCAD完成：模板长 75mm，宽 25mm, 均匀分布 3 X 14个直径为 4mm的微孔作为支架模型，每个微孔的中心距离为 1000 m。激光雕刻机的主要加工参数为：切割能量 100%，切割次数 2，切割线速度 10%。见图 2-Α。 2. 化学交联法制备水凝胶 A polymethyl methacrylate (PMMA) plate having a thickness of 0.5 mm, 1 mm, 2 mm, and 3 mm was formed by a Rayjet laser engraving machine to form a mold. The template design is done by the software AutoCAD: The template is 75mm long and 25mm wide. It is evenly distributed with 3 X 14 micropores with a diameter of 4mm as the support model. The center distance of each micro hole is 1000 m. The main processing parameters of the laser engraving machine are: cutting energy 100%, cutting times 2, cutting line speed 10%. See Figure 2-Α. 2. Preparation of hydrogel by chemical crosslinking

本发明化学交联法制备水凝胶所配制的预聚物溶液 B 的一大特点就是透明剂的添力口，从而满足生物学、药学、医学等领域对三维微环境实施无标记成像研究的要求。可用的透明剂主要为多元醇类，如乙二醇， 1,3-丁二醇， 1,2,4-丁三醇，丙三醇， 1,2-丙二醇， 1,3-丙二醇，季戊四醇，顺 -1,2-环戊二醇，丁四醇，戊五醇等。所述透明剂多元醇在最终的预聚物溶液 B中的体积百分比为 0.01%-100%。下述将以 1,2,4-丁三醇作为透明剂，阐述所述预聚物溶液 B的配制及水凝胶的制备方法：将 10% (w/v) 聚乙二醇二丙烯酸酯于 60°C条件下溶解于 1,2,4-丁三醇与水（体积比 60/40 ) 的混合溶液中。在冰盒上操作，加入 0.05% (w/v) 过硫酸铵的和 0.5% (w/v) Ν,Ν,Ν',Ν'-四甲基二乙胺得到预聚物溶液 Β (上述各物质的浓度均为在预聚物溶液 Β中的终浓度）。将此预聚物溶液 Β 滴加到 ΡΜΜΑ模具的微孔中，在室温条件下溶液逐渐化学交联为透明的水凝胶。实验中，同时设置不加入透明剂 1,2,4-丁三醇的对照。 The pre-polymer solution B prepared by preparing the hydrogel by the chemical cross-linking method of the invention is characterized by the addition of a transparent agent, thereby satisfying the research of label-free imaging for the three-dimensional microenvironment in the fields of biology, pharmacy and medicine. Claim. Useful clearing agents are mainly polyols such as ethylene glycol, 1,3-butanediol, 1,2,4-butanetriol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol. , cis-1,2-cyclopentanediol, tetrabutyl alcohol, pentaerythritol, and the like. The volume percentage of the clearing agent polyol in the final prepolymer solution B is from 0.01% to 100%. The preparation of the prepolymer solution B and the preparation method of the hydrogel will be described below using 1,2,4-butanetriol as a transparent agent: 10% (w/v) polyethylene glycol diacrylate It was dissolved in a mixed solution of 1,2,4-butanetriol and water (60/40 by volume) at 60 °C. Operate on an ice box, add 0.05% (w/v) ammonium persulfate and 0.5% (w/v) Ν, Ν, Ν', Ν'-tetramethyldiethylamine to obtain a prepolymer solution Β (above The concentration of each substance is the final concentration in the prepolymer solution enthalpy). The prepolymer solution was added dropwise to the micropores of the crucible mold, and the solution was gradually chemically crosslinked into a transparent hydrogel at room temperature. In the experiment, a control in which the clearing agent 1,2,4-butanetriol was not added was set at the same time.

3. 海绵支架的制备 3. Preparation of sponge scaffold

采用冷冻干燥法将上述水凝胶制备成多孔支架。具体过程如下：将上述载有水凝胶的模具浸于超纯水中除去未交联的单体、 1,2,4-丁三醇等杂质，换 4-5次水。之后，将其在 -20°C条件下冷冻 4-5h，再转入冷冻干燥机（-50°C， 20pa)干燥 12小时，得到白色多孔海绵支架。按照上述方法，制备得到海绵支架，其实物图和电镜图见图 2-B。所得海绵支架的体积大小为 6.26mm³、 12.52 mm³、 25.04mm³、 37.56mm³;孔径大小 ΙΟμηι -150μηΐ; 孔间距 Ιμηι -999 μ m; 孔隙率为 94.2%，连通性良好；吸水量为理论体积的 10-15倍。 The above hydrogel was prepared into a porous scaffold by freeze drying. The specific process is as follows: The above hydrogel-loaded mold is immersed in ultrapure water to remove impurities such as uncrosslinked monomers and 1,2,4-butanetriol, and replaced with water for 4-5 times. Thereafter, it was frozen at -20 ° C for 4-5 h, and then transferred to a freeze dryer (-50 ° C, 20 Pa) for 12 hours to obtain a white porous sponge scaffold. According to the above method, a sponge scaffold is prepared, and the physical map and the electron microscope image are shown in Fig. 2-B. Sponges resulting size of the volume of ^{^{6.26mm 3, 12.52 mm 3, 25.04mm}} 3, 37.56mm 3; pore size ΙΟμηι -150μηΐ; hole pitch Ιμηι -999 μ m; porosity of 94.2%, good communication; water absorption 10-15 times the theoretical volume.

(二）光交联法制备图案化透明海绵支架 (2) Preparation of patterned transparent sponge scaffold by photocrosslinking

1. 光掩模的设计与打印 1. Photomask design and printing

采用 AutoCAD绘图软件设计光掩模。设计尺寸：光掩模大小为 76.2mmx25.4mm，根据需求自由设计图案。例如，图 2中 C设计尺寸：透光孔径 D=lmm，孔间距 W=2mm。图 2中 D设计尺寸：透光环内径 D _ή=1200μηι, 外径 D外 =2000、 2560、 3020、 3420、 3780、 4100、 4400、 4680μηι, 环间距 ¥=6000μηι。在印刷厂（清华大学印刷厂）打印胶片光掩模。 The photomask was designed using AutoCAD drawing software. Design size: The photomask size is 76.2mmx25.4mm, and the pattern can be freely designed according to requirements. For example, the design dimensions of C in Figure 2 are: light transmission aperture D = 1 mm, hole spacing W = 2 mm. In Figure 2, the design dimensions of D: the inner diameter of the transparent ring D _ή = 1200μηι, the outer diameter D outside = 2000, 2560, 3020, 3420, 3780, 4100, 4400, 4680μηι, ring spacing ¥ = 6000μηι. Printed a film photomask at a printing house (Tsinghua University Printing Factory).

2. 可光交联的预聚物溶液 Α的配制 2. Photocrosslinkable prepolymer solution

本发明配制可光交联的预聚物溶液 A的一大特点就是透明剂的添加，从而满足生物学、药学、医学等领域对三维微环境实施无标记成像研究的要求。可用的透明剂主要为多元醇类，如乙二醇， 1,3-丁二醇， 1,2,4-丁三醇，丙三醇， 1,2-丙二醇， 1,3-丙二醇，季戊四醇，顺 -1,2-环戊二醇，丁四醇，戊五醇等。所述透明剂多元醇在最终的可光交联的预聚物溶液 A中的体积百分比为 0.01%-100%。下述将以 1,2,4-丁三醇作为透明剂，阐述所述可光交联的预聚物溶液 A的配制方法：将 10% (w/v)聚乙二醇二丙烯酸酯和 0.5% (w/v) 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮于 60°C条件下溶解于 1,2,4-丁三醇与水（体积比 60/40) 的混合溶液中，冷却后得到可光交联的预聚物溶液 A (上述各物质的浓度均为在可光交联的预聚物溶液 A中的终浓度）。混匀贮存于 4°C环境中备用。实验中，同时设置不加入透明剂 1,2,4-丁三醇的对照。 A major feature of the photocrosslinkable prepolymer solution A of the present invention is the addition of a clearing agent, thereby meeting the requirements for the implementation of label-free imaging in a three-dimensional microenvironment in the fields of biology, pharmacy, and medicine. Useful clearing agents are mainly polyols such as ethylene glycol, 1,3-butanediol, 1,2,4-butanetriol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol. , cis-1,2-cyclopentanediol, tetrabutyl alcohol, pentaerythritol, and the like. The volume percentage of the clearing agent polyol in the final photocrosslinkable prepolymer solution A is from 0.01% to 100%. The preparation method of the photocrosslinkable prepolymer solution A will be described below using 1,2,4-butanetriol as a transparent agent: 10% (w/v) polyethylene glycol diacrylate and 0.5% (w/v) 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone dissolved in 1,2,4-butanetriol and water at 60 ° C (volume ratio In the mixed solution of 60/40), the photocrosslinkable prepolymer solution A was obtained after cooling (the concentration of each of the above substances was the final concentration in the photocrosslinkable prepolymer solution A). Mix and store in 4 ° C environment for use. In the experiment, a control in which the clearing agent 1,2,4-butanetriol was not added was simultaneously set.

3. 载玻片的包被采用化学修饰法分别将载玻片包被上十八烷基三氯硅烷（OTS ) (百灵威科技有限公司）和甲基丙烯酸 3- (三甲氧基硅烷)丙酯（TMP) ， OTS暴露端十八烷基将载玻片改性为疏水性，可以使液体聚集成一定高度， TMP暴露端双键可以与聚乙二醇二丙烯酸酯的双键反应使聚合物固定在载玻片上。具体包被方法如下： 1) 将载玻片放到染色架上，浸泡于洗洁***溶液中，超声 20分钟； 2) 将载玻片染色架浸于 10%的氢氧化钠溶液 60分钟； 3) 再浸于超纯水中 30分钟，烘干； 4) 室温下，将载玻片染色架浸于含有 5% (V/V) OTS的正己烷溶液中 30分钟，或含有 1% (V/V) TMP的乙醇（含 3% 乙酸）中 3分钟； 5) 将包被上 OTS的载玻片在甲苯中浸泡 10分钟，将包被上 TMP 的载玻片在无水乙醇中浸泡 10分钟； 6) 最后将载玻片烘干，储存于 4°C冰箱中备用。 3. Slides for slides The slides were coated with octadecyltrichlorosilane (OTS) (Belling Technology Co., Ltd.) and 3-(trimethoxysilane) propyl methacrylate (TMP) by chemical modification, and the OTS exposed end ten The octaalkyl group modifies the slide to hydrophobicity, allowing the liquid to aggregate to a certain height. The double bond at the exposed end of the TMP can react with the double bond of the polyethylene glycol diacrylate to immobilize the polymer on the slide. The specific coating method is as follows: 1) Place the slide on the staining rack, soak in the aqueous detergent solution, and sonicate for 20 minutes; 2) immerse the slide staining rack in 10% sodium hydroxide solution for 60 minutes; 3) Re-immersion in ultrapure water for 30 minutes, drying; 4) Immerse the slide staining rack in a solution of 5% (V/V) OTS in n-hexane for 30 minutes at room temperature, or contain 1% ( V/V) TMP in ethanol (containing 3% acetic acid) for 3 minutes; 5) Soak the OTS-coated slide in toluene for 10 minutes, soak the TMP-coated slide in absolute ethanol 10 minutes; 6) Finally, the slides are dried and stored in a refrigerator at 4 °C for use.

4. 紫外光交联过程 4. UV crosslinking process

按照图 1-B-5 (影印石版术示意图）所示构造图，将上述步骤 1、 2、 3制备好的光掩模、可光交联的预聚物溶液、包被好的载玻片，以及盖玻片组合起来，将其暴露在紫外光下形成图案状水凝胶，支架高度为 2 个盖玻片， 300μηι。紫外光源为加拿大 Omnicure S2000紫外交联仪；具体紫外交联参数为：紫外光强度 20mW/cm²，光照时间 8.5s。 According to the configuration diagram shown in Figure 1-B-5 (photographed stone diagram), the photomask prepared by the above steps 1, 2, 3, the photocrosslinkable prepolymer solution, and the coated glass slide And the coverslips are combined and exposed to ultraviolet light to form a patterned hydrogel with a height of 2 coverslips, 300 μm. The ultraviolet light source is the Canadian Omnicure S2000 UV cross-linking instrument; the specific UV cross-linking parameters are: ultraviolet light intensity 20mW/cm ² , illumination time 8.5s.

5、图案化海绵支架的制备 5. Preparation of patterned sponge scaffold

采用冷冻干燥法将水凝胶制备成多孔支架。具体过程如下：将上述步骤 4交联后的组合轻轻拆卸，取固定有水凝胶的载玻片（即支撑所述海绵支架或所述海绵支架阵列的基底 B )浸于超纯水中除去未交联的单体、 1,2,4-丁三醇和杂质，换 4-5次水。之后，将其在 -20°C条件下冷冻 4-5h，再转入冷冻干燥机（-50°C， 20pa)干燥 12小时，得到白色三维图案化海绵支架。按照上述方法，制备得到不同大小的海绵支架，其实物图和电镜图见图 2中 C和 D。其中，图 2中 C的海绵支架体积大小均为 0. 2355μ1。图 2中 D的海绵支架体积大小分别为 0.603μ1、 1.204μ1、 1.809μ1、 2.415μ1、 3.026μ1、 3.620μ1、 4.22μ1、 4.82μ1。图 2中 C所示的海绵支架的孔径大小均在 10μηι-125μηι; 孔间距 Ιμηι -999μηι, 孔径分布百分比率均为（见图 2中 Ε) ： 10μηι-30μηι的小孔占 17.74%， 30μηι-50μηι的小孔占 43.55%， 50μηι-80μηι的小孔占 32.56%，孔径大小 80μηι-125μηι的小孔占 6.45%; 孔隙率为 93.3%，连通性良好；吸水量为理论体积的 3.5-5倍。图 2中 D所示的海绵支架的孔径大小均在 1μηι-85μηι_; 孔间距 Ιμηι -999μηι, 孔径分布百分比率均为（见图 2 中 F) ： 1μηι-10μηι的小孔占 5.33%， 10μηι-20μηι的小孔占 27.83%， 20μηι-30μηι的小孔占 35.00%，孔径大小 30μηι-40μηι的小孔占 20.50%，孔径大小 40μηι-85μηι的小孔占 11.33%；孔隙率为 82.4%，连通性良好；吸水量为理论体积的 1-2倍。 The hydrogel was prepared into a porous scaffold by freeze drying. The specific process is as follows: The combination of the cross-linking of the above step 4 is gently disassembled, and the hydrogel-fixed glass slide (ie, the substrate B supporting the sponge scaffold or the sponge scaffold array) is immersed in ultrapure water. The uncrosslinked monomer, 1,2,4-butanetriol and impurities were removed and replaced with 4-5 times of water. Thereafter, it was frozen at -20 ° C for 4-5 h, and then transferred to a freeze dryer (-50 ° C, 20 Pa) for 12 hours to obtain a white three-dimensional patterned sponge scaffold. According to the above method, different sizes of sponge scaffolds were prepared. The physical map and the electron micrograph are shown in C and D in Fig. 2. 2355μ1。 The size of the sponge scaffold is 0. 2355μ1. The volume of the sponge scaffold of D in Fig. 2 is 0.603μ1, 1.204μ1, 1.809μ1, 2.415μ1, 3.026μ1, 3.620μ1, 4.22μ1, 4.82μ1, respectively. The pore size of the sponge scaffold shown in Figure 2C is 10μηι-125μηι; the pore spacing is Ιμηι -999μηι, and the percentage distribution of pore size distribution is (see Fig. 2): the pores of 10μηι-30μηι account for 17.74%, 30μηι- The pores of 50μηι accounted for 43.55%, the pores of 50μηι-80μηι accounted for 32.56%, the pores with pore size of 80μηι-125μηι accounted for 6.45%, the porosity was 93.3%, the connectivity was good, and the water absorption was 3.5-5 times of the theoretical volume. . The pore size of the sponge scaffold shown in D in Fig. 2 is 1μηι-85μηι _{; the} pore spacing is Ιμηι -999μηι, and the percentage distribution of pore size distribution is (see F in Fig. 2): the pores of 1μηι-10μηι account for 5.33%, 10μηι- The pores of 20μηι accounted for 27.83%, the pores of 20μηι-30μηι accounted for 35.00%, the pores with pore size of 30μηι-40μηι accounted for 20.50%, and the pores with pore size of 40μηι-85μηι accounted for 11.33%; porosity was 82.4%, connectivity Good; water absorption is 1-2 times the theoretical volume.

实施例 2、海绵支架自动装载分子、材料、细胞 Example 2. Sponge stent automatically loads molecules, materials, cells

本案例实现分子、材料和细胞的自动装载方法为传统滴加方法（图 1中 B-1 ) 。传统滴加方法是指将样品液体直接滴加在支架表面或旁侧，样品液自动被吸入支架内部。所述样品液可为小分子化合物、药物、核酸、蛋白、细胞外基质成分、高分子材料、微珠、真核细胞、原核细胞、病毒、微生物中的任一种或任几种的混合物。 In this case, the automatic loading method for molecules, materials and cells is the traditional dropping method (B-1 in Fig. 1). The conventional dropping method means that the sample liquid is directly dropped on the surface or the side of the stent, and the sample liquid is automatically sucked into the inside of the stent. The sample solution may be any one or a mixture of a small molecule compound, a drug, a nucleic acid, a protein, an extracellular matrix component, a polymer material, a microbead, a eukaryotic cell, a prokaryotic cell, a virus, a microorganism, or any one of several.

本实施例选用 ρΗ指示剂酚红作为样品液中分子样品的示例（图 3中 Α和 B)、 lmg/ml 的明胶作为样品液中材料样品的示例（图 3中 C和 D) 、 5 X 10⁶个 /ml的 HeLa细胞（中国医学科学院基础医学研究所基础医学细胞中心， 3111C0001CCC000011 )作为细胞样品（图 3中 E) 自动装载进入实施例 1光交联制备好的三维图案化 "海绵支架" 中。其中，图 3中 A的圆柱形海绵支架是按照实施例 1所述光交联法制备而来的，圆柱的直径为 ΙΟΟΟμηι; 海绵支架的高度为 300μηι_; 其海绵支架的孔径大小 1μηι-85μηι_; 孔间距 1μηι-999μηΐ; 孔隙率为 82.4%，连通性良好；吸水量为理论体积的 1-2倍；该海绵支架阵列共由 64个海绵支架组成。图 3-C和 3-Ε中的正六边形和长方形海绵支架是按照实施例 1所述光交联法制备而来的，正六边形的边长为 500μηι，长方形的长为 ΙΟΟΟμηι, 宽为 500μηι_; 海绵支架的高度为 300μηι_; 海绵支架的孔径大小 10μηι-125μηι_; 孔间距 1μηι-999μηΐ; 孔隙率为 93.3%，连通性良好；吸水量为理论体积的 3.5-5倍；这两海绵支架阵列均共由 16个海绵支架组成。用荧光显微镜拍摄记录海绵支架自动装载 5χ10⁶ 个 /ml的荧光微球（直径 ΙΟμηι) 液体的动态过程（图 3中 F) 。其中， F-1为 1.5秒， F-2为 2.5秒， F-3为 3.5秒， F-4为 4.5秒， F-5为 5.5秒， F-6为 7秒， F-7为 8.5秒， F-8为 11秒， F-9为 13秒。吸水量为 5μ1的海绵支架完成自动装载过程耗时 13秒。 In this embodiment, the ρΗ indicator phenol red is used as an example of a molecular sample in the sample liquid (Α and B in Fig. 3), and lmg/ml gelatin is used as an example of a material sample in the sample liquid (C and D in Fig. 3), 5 X 10 ⁶ / ml of HeLa cells (medium The Basic Medical Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, 3111C0001CCC000011) was automatically loaded into the three-dimensional patterned "sponge holder" prepared by the photocrosslinking of Example 1 as a cell sample (E in Fig. 3). The cylindrical sponge scaffold of A in FIG. 3 is prepared by the photocrosslinking method according to Embodiment 1, wherein the diameter of the cylinder is ΙΟΟΟμηι; the height of the sponge scaffold is 300 μm _; and the pore size of the sponge scaffold is 1 μηι-85 μηι _; The pore spacing is 1μηι-999μηΐ; the porosity is 82.4%, the connectivity is good; the water absorption is 1-2 times of the theoretical volume; the sponge stent array is composed of 64 sponge stents. The regular hexagonal and rectangular sponge holders in Figures 3-C and 3-Ε were prepared by the photocrosslinking method described in Example 1. The sides of the regular hexagon were 500 μm, and the length of the rectangle was ΙΟΟΟμηι, and the width was 500μηι _; sponge holder height is 300μηι _; sponge holder pore size 10μηι-125μηι _; pore spacing 1μηι-999μηΐ; porosity 93.3%, good connectivity; water absorption is 3.5-5 times the theoretical volume; They are all composed of 16 sponge scaffolds. The dynamic process of automatically loading 5 χ ^{10 6} /ml of fluorescent microspheres (diameter ημηι) with a fluorescent microscope was recorded by fluorescence microscopy (F in Fig. 3). Among them, F-1 is 1.5 seconds, F-2 is 2.5 seconds, F-3 is 3.5 seconds, F-4 is 4.5 seconds, F-5 is 5.5 seconds, F-6 is 7 seconds, F-7 is 8.5 seconds. F-8 is 11 seconds and F-9 is 13 seconds. The sponge holder with a water absorption of 5 μ1 completed the automatic loading process and took 13 seconds.

实施例 3、海绵支架透明光学性能测试 Example 3: Transparent optical performance test of sponge scaffold

本实施例选用文字观察和透明度测量来检测海绵支架的透明光学性能。 In this embodiment, text observation and transparency measurement are used to detect the transparent optical properties of the sponge holder.

将按照实施例 1中化学交联法和光交联法制备的海绵支架（图 2中 Β，图 3中 C) 浸入水中，置于 37 °C培养箱中 2-3天，除去支架内部气泡。将其分别置于载玻片上，透过支架观察文字（图 4中 A， C和 D ) 。通过对比观察，本发明方法制备的海绵支架再次吸水后光学性能良好，可以清晰看到载玻片下的文字。没有加入透明剂的样品光学性能较差，无法清晰看到载玻片下文字。将不同体积比（0%， 10% , 30% , 60% ) 透明剂 1 ,2,4-丁三醇和不同高度的体积比为 60%透明剂 1 ,2,4-丁三醇的样品分别放在载玻片上，用酶标仪检测其透光度（同时以 1cm玻璃作为对照），得到透明海绵支架的透明光学性能定量图（图 4中 E和 F)。根据此定量图，可知：透明海绵支架的透明度随透明剂 1,2,4- 丁三醇的体积比的增大而增大，随透明海绵支架的高度的增大而减小。 The sponge scaffold (Fig. 2, 3, Fig. 3, C) prepared according to the chemical cross-linking method and the photocrosslinking method of Example 1 was immersed in water and placed in a 37 ° C incubator for 2-3 days to remove air bubbles inside the scaffold. Place them on a glass slide and observe the text through the holder (A, C and D in Figure 4). By contrast observation, the sponge scaffold prepared by the method of the present invention has good optical properties after repeated water absorption, and the text under the slide glass can be clearly seen. Samples without a clearing agent had poor optical properties and could not clearly see the text under the slide. Different volume ratios (0%, 10%, 30%, 60%) of the transparent agent 1,2,4-butanetriol and the volume ratio of different heights of 60% of the transparent agent 1,2,4-butanetriol were respectively It was placed on a glass slide, and its transmittance was measured by a microplate reader (while using 1 cm of glass as a control) to obtain a quantitative optical property quantitative view of the transparent sponge scaffold (E and F in Fig. 4). According to this quantitative chart, it is understood that the transparency of the transparent sponge stent increases as the volume ratio of the transparent agent 1,2,4-butanetriol increases, and decreases as the height of the transparent sponge stent increases.

实施例 4、观察记录细胞在透明海绵支架中生长 Example 4. Observation of recorded cells growing in a transparent sponge scaffold

本实施例选用 HeLa细胞作为案例观察记录其在透明海绵支架中的生长。将 5 X 10⁶ 个 /ml的 HeLa细胞悬浮液滴加在海绵支架上，并培养于 37 °C、 5%C0₂、饱和湿度培养箱中，培养液为添加 10% ( v/v) FBS禾 B 1% ( v/v)双抗（青链霉素、链霉素）的 DMEM 培养液，每两天换液一次。普通白光显微镜观察记录 HeLa细胞随时间在透明海绵支架 (实施例 1中化学交联法制备得到的海绵支架）中逐渐生长形成肿瘤微球（图 5中 A-1 至 A-5 ) 。其中， A-1至 A-5分别为细胞种植 0h、 4h、 26h、 50h、 lOOh后的记录图片。通过观察比较，可清晰观察到 HeLa细胞逐渐生长形成肿瘤微球，且肿瘤微球随时间逐渐增大。（见箭头标记）。换液培养六天后，进行 PBS洗涤、 4%多聚甲醛固定 lOmiiu PBS洗漆、 0.5% ( v/v) triton通透 5min、 PBS洗涤、 100nM罗丹明（Rhodamine) 室温染色 30min、 PBS洗涤、 100nM DAPI 37 °C染色 10min、 PBS洗涤、覆盖抗荧光猝灭剂等一系列步骤将支架内的肿瘤微球实现荧光染色。图 5中 B为荧光显微镜观察肿瘤微球图片，图 5中 C为扫描电镜观察肿瘤微球，图 5中 D为激光共聚焦显微镜观察肿瘤微球图片（细胞核：蓝色 -DAPI, 细胞骨架：红色 -rhodamine ) ，进一步证明 HeLa细胞在三维环境中的生长状态为三维立体生长，不同于传统两维环境中单细胞层生长。 In this example, HeLa cells were selected as a case observation to record their growth in a transparent sponge scaffold. 5 X 10 ⁶ /ml HeLa cell suspension was added to the sponge scaffold and cultured in a 37 ° C, 5% CO ₂ , saturated humidity incubator. The culture medium was added with 10% (v/v) FBS. DMEM medium containing 1% (v/v) double antibody (cyanin, streptomycin), changed every two days. Ordinary white light microscopy observation showed that HeLa cells gradually grew to form tumor microspheres in a transparent sponge scaffold (the sponge scaffold prepared by chemical cross-linking in Example 1) (A-1 to A-5 in Fig. 5). Among them, A-1 to A-5 are recorded pictures after cell planting for 0h, 4h, 26h, 50h, lOOh. By observation and comparison, it can be clearly observed that HeLa cells gradually grow to form tumor microspheres, and the tumor microspheres gradually increase with time. (see arrow mark). After 6 days of liquid exchange culture, PBS washing, 4% paraformaldehyde fixed lOmiiu PBS wash, 0.5% (v/v) triton permeation for 5 min, PBS washing, 100 nM rhodamine (Rhodamine) room temperature staining for 30 min, PBS washing, 100 nM The DAPI was stained at 37 °C for 10 min, washed with PBS, covered with anti-fluorescent quencher, and the like, and the tumor microspheres in the scaffold were fluorescently stained. In Fig. 5, B is a microscopic picture of the tumor microsphere. In Fig. 5, C is a scanning electron microscope to observe the tumor microsphere. In Fig. 5, D is a laser confocal microscope to observe the tumor microsphere picture (nucleus: blue-DAPI, cytoskeleton: Red-rhodamine ), further demonstrating that HeLa cells are The growth state in the three-dimensional environment is three-dimensional growth, which is different from the single-cell layer growth in the traditional two-dimensional environment.

实施例 5、海绵支架中细胞的传代培养 Example 5: Subculture of cells in a sponge scaffold

本实施例选用 HeLa细胞作为案例进行传代培养。将实施例 4透明支架中的 HeLa 肿瘤微球（图 6中 A) 经过 0.25%胰酶消化 2min、培养液洗涤 3-4次，得到 HeLa细胞悬浮液（图 6中 B) 。经过 0.2% (w/v) Calcein-AM ( sigma, C1359, 染色操作参见说明书）和 0.3% (w/v) PI染液（死细胞：红色 -PI，活细胞：绿色 -Calcein-AM)染色 15min 后，荧光显微镜观察细胞保持接近 100%的存活率（图 6中 C-1和 C-2) 。其中 C-1为低倍镜 2X观察图片， C-2为高倍镜 10 X观察图片。 In this example, HeLa cells were used as a case for subculture. The HeLa tumor microspheres (Fig. 6 A) in the transparent stent of Example 4 were subjected to 0.25% trypsin digestion for 2 min, and the culture solution was washed 3-4 times to obtain a HeLa cell suspension (B in Fig. 6). After 0.2% (w/v) Calcein-AM (sigma, C1359, staining operation see instructions) and 0.3% (w/v) PI stain (dead cells: red-PI, living cells: green-Calcein-AM) After 15 min, the cells were observed to maintain near 100% survival by fluorescence microscopy (C-1 and C-2 in Figure 6). Among them, C-1 is the low magnification lens 2X observation picture, C-2 is the high magnification lens 10 X observation picture.

实施例 6、液体薄层法将药物分子、材料自动装载于高通量、图案化海绵支架阵列本案例实现分子、材料的高通量、图案化自动装载方法为液体薄层法（图 1中 B-4)。液体薄层法是指将图案化海绵支架阵列覆盖在液体薄层上或将液体薄层覆盖在图案化海绵支架阵列上，利用海绵的吸附作用和液体薄层的易流动分离性，自动将液体吸进海绵支架内，从而实现样品液的三维微尺度装载。下述内容涉及用于构建三维微环境的装置的基底 A的制作、所述基底 A上疏水边框的制作，以及利用液体薄层法实现分子、材料的高通量、图案化自动装载的操作步骤。具体如下： Example 6. Liquid thin layer method automatically loading drug molecules and materials into a high-flux, patterned sponge scaffold array. In this case, a high-throughput, patterned auto-loading method for molecules and materials is a liquid thin layer method (in FIG. 1). B-4). The liquid thin layer method refers to covering a patterned sponge scaffold array on a thin liquid layer or covering a thin layer of liquid on the patterned sponge scaffold array, and utilizing the adsorption of the sponge and the easy flow separation of the liquid thin layer, automatically introducing the liquid It is sucked into the sponge holder to achieve three-dimensional micro-scale loading of the sample solution. The following relates to the fabrication of the substrate A for the device for constructing the three-dimensional microenvironment, the fabrication of the hydrophobic frame on the substrate A, and the high-throughput, patterned automatic loading of the molecules and materials using the liquid thin layer method. . details as follows:

1. 等离子清洗法修饰亲水性基底载玻片 1. Plasma cleaning method to modify hydrophilic substrate slides

采用 HPDC 基础型等离子清洗***将载玻片改性为亲水性基底，即制作用于构建三维微环境的装置的基底 A。具体过程如下： 1 ) 将载玻片放置入清洗机的舱内，打开等离子清洗***中的真空油泵，抽真空 5分钟； 2)待舱内产生紫色辉光时，计时清洗 1 分钟；改性效果见图 7中 A-1和 A-2 (A-1清洗前水的接触角， A-2清洗后水的接触角）。液体可在基底上形成薄层。 The slides were modified to a hydrophilic substrate using an HPDC basic plasma cleaning system, i.e., substrate A for making a device for constructing a three-dimensional microenvironment. The specific process is as follows: 1) Place the slide into the cabin of the washing machine, open the vacuum oil pump in the plasma cleaning system, and vacuum for 5 minutes; 2) When the purple glow is generated in the cabin, the time is cleaned for 1 minute; The effect is shown in Figure 7 for A-1 and A-2 (A-1 contact angle of water before washing, A-2 contact angle of water after washing). The liquid can form a thin layer on the substrate.

2. 化学法修饰疏水性边框 2. Chemical modification of hydrophobic borders

1 ) 按照高通量海绵支架阵列载玻片（图 7中 B的多边形的海绵支架阵列是按照实施例 1所述光交联法制备而来的，多边形的外直径为 ΙΟΟΟμηι;海绵支架的高度为 300μηι_; 其海绵支架的孔径大小 1μηι-85μηι_; 孔间距 1μηι-999μηι_; 孔隙率为 82.4%，连通性良好；吸水量为理论体积的 1-2倍；该海绵支架阵列共由 192个海绵支架组成。）的设计，用激光雕刻机（Rajet)切割厚度为 0.5mm的 PMMA平板，得到与海绵支架阵列区域划分1) According to the high-throughput sponge scaffold array slide (the polygonal sponge scaffold array of B in Fig. 7 is prepared according to the photocrosslinking method described in Example 1, the outer diameter of the polygon is ΙΟΟΟμηι; the height of the sponge scaffold It is 300μηι _; its sponge scaffold has a pore size of 1μηι-85μηι _; pore spacing is 1μηι-999μηι _; porosity is 82.4%, connectivity is good; water absorption is 1-2 times theoretical volume; the sponge scaffold array is composed of 192 sponge scaffolds Composition.) The laser engraving machine (Rajet) cuts the PMMA plate with a thickness of 0.5mm to obtain the area division with the sponge stent array.

(2X 6) 重合的 PMMA边界模具； 2) 沿 PMMA模具的边界均匀涂抹 50μ1 1% (ν/ν) OTS/正己烷混合溶液，并放在匀胶机（Mycro) 上以 3000r/min的速度旋匀 30s; 3) 将修饰后的 PMMA模具对齐按压在经上述等离子清洗法处理后的亲水性载玻片（基底 A) 上 4s; 4)在步骤 3载玻片（基底 A)上一一对应海绵支架阵列区域滴加 1C L不同颜色的染料溶液（葡萄紫，亮蓝，果绿，胭脂红，日落黄，苋菜红），染料溶液在每个区域自动分散成均匀液体薄层（图 7中 C) _; (2X 6) coincident PMMA boundary mold; 2) uniformly spread 50μ1 1% (ν/ν) OTS/n-hexane mixed solution along the boundary of PMMA mold and place it on the homogenizer (Mycro) at a speed of 3000r/min. Rotate for 30 s; 3) Align the modified PMMA mold on the hydrophilic slide (substrate A) treated by the above plasma cleaning method for 4 s; 4) On step 3 (slide A) A 1C L dye solution of different colors (V. Violet, Brilliant Blue, Fruit Green, Carmine, Sunset Yellow, Amaranth) is added to the corresponding sponge scaffold array area, and the dye solution is automatically dispersed into a uniform liquid thin layer in each area (Fig. 7 in C) _;

3. 液体薄层法自动装载分子、材料进入高通量、图案化海绵支架 3. Liquid thin layer method automatically loads molecules and materials into high-throughput, patterned sponge scaffolds

将高通量的海绵支架阵列载玻片（基底 B及固定其上的海绵支架阵列）（图 7中 B 所示海绵支架阵列）按照区域划分平行正对轻轻覆盖在上述载有不同颜色染料液体薄层 (液体薄层厚度为 10微米）的载玻片（基底 A) 上，使海绵支架每个阵列区域（2X 6) 同时接触区域液体薄层，区域液体自动被海绵支架吸收，从而达到高通量并行自动装载的目的（图 7中 D) 。 Place a high-throughput sponge scaffold array slide (substrate B and an array of sponge scaffolds attached thereto) (the sponge scaffold array shown in Figure 7) in parallel according to the area and gently cover the dyes containing different colors. On a thin glass slide (substrate A) with a thin liquid layer (10 μm thick), each array area (2× 6) of the sponge holder is simultaneously contacted with a thin layer of liquid, and the liquid in the area is automatically absorbed by the sponge holder. High-throughput parallel autoloading The purpose (D in Figure 7).

按照上述三个步骤采用液体薄层法将 Doxorubicin药物（红色荧光）和荧光微球（绿色，直径 ΙΟμηι) 同时自动装载进入图案化海绵支架中（图 7中 Ε) 。实现多种液体的简易同步装载。图 7中 Ε中的清华百年校庆图标状海绵支架是按照实施例 1所述光交联法制备而来的，海绵支架的高度为 300μηι_; 海绵支架的孔径大小 10μηι-125μηι_; 孔间距 1μηι-999μηΐ; 孔隙率为 93.3%，连通性良好；吸水量为理论体积的 3.5-5倍。 The Doxorubicin drug (red fluorescence) and the fluorescent microsphere (green, diameter ΙΟμηι) were simultaneously loaded into the patterned sponge scaffold using the liquid thin layer method in the above three steps (Fig. 7 Ε). Achieve simple simultaneous loading of multiple liquids. The Tsinghua Centennial Icon-like Sponge Scaffold in Figure 7 was prepared according to the photocrosslinking method described in Example 1. The height of the sponge scaffold was 300 μηι _; the pore size of the sponge scaffold was 10 μηι-125 μηι _{; the} pore spacing was 1 μηι-999 μηΐ The porosity is 93.3%, and the connectivity is good; the water absorption is 3.5-5 times the theoretical volume.

实施例 7、液体薄层法自动装载 HeLa细胞种植入高通量海绵支架阵列 Example 7. Automatic loading of liquid thin layer method HeLa cells were planted into a high-throughput sponge scaffold array

本实施例先采用 Calcein-AM试剂（sigma, C1359) 检测高通量海绵支架阵列能否实现对 HeLa细胞的自动装载，接着选用 Cell Titer-Blue试剂（Promega, G8080) 研究液体薄层法高通量自动装载 HeLa细胞的效率。 In this example, Calcein-AM reagent (sigma, C1359) was used to detect whether the high-throughput sponge scaffold array can automatically load HeLa cells. Then, Cell Titer-Blue reagent (Promega, G8080) was used to study the liquid thin layer method Qualcomm. The efficiency of loading HeLa cells automatically.

首先，将 HeLa细胞悬浮液轻轻吹打均匀，并用 Calcein-AM (其使用方法参照试剂说明书）处理细胞，取 200μ1不同浓度的悬浮液分别均匀平铺在不同的亲水性载玻片基底（基底 Α)上，轻轻将高通量海绵支架陈列载玻片（基底 Β及固定其上的海绵支架阵列，图 8中 Α的圆环形海绵支架是按照实施例 1所述光交联法制备而来的，圆环的内径为 2400μηι，外径为 3400μηι _; 海绵支架的高度为 300μηι _; 海绵支架的孔径大小 10μηι-125μηΐ; 孔间距 1μηι-999μηι; 孔隙率为 93.3%，连通性良好；吸水量为理论体积的 3.5-5倍；该海绵支架阵列共由 24个海绵支架组成。）覆盖在细胞液体薄层（液体薄层厚度为 50微米）上，待细胞溶液分散进入海绵支架内部后，轻轻移去支架阵列载玻片，实现 HeLa细胞的高通量三维装载（图 8中 A， HeLa细胞：绿色 -Calcein-AM) 。不同细胞浓度液体薄层装载后的效果图见图 8中 B-1至 B-3 (B-1 : 2X 10⁶个 /ml， B-2: 4X 10⁵个 /ml， B-3: 8 X 10⁴个 /ml) 。采用激光共聚焦显微镜扫描海绵支架内部的细胞分布，见图 8中 C-1至 C-3 (C-l : 2X 10⁶个 /ml， C-2: 4X 10⁵个 /ml， C-3: 8 X 10⁴个 /ml) 。结果表明，随着细胞浓度的提高，自动装载进入海绵支架的细胞越多，绿色荧光强度越强，细胞三维分布越密集，特别是图 8中 B-1和 C-l。这说明上述高通量海绵支架陈列能够通过液体薄层法实现对 HeLa细胞的自动装载。 First, the HeLa cell suspension was gently pipetted evenly, and the cells were treated with Calcein-AM (the method of use is referred to the reagent instructions), and 200 μl suspensions of different concentrations were uniformly spread on different hydrophilic slide substrates (base). On the Α), gently place the high-throughput sponge scaffold on the slide glass (the substrate Β and the sponge holder array fixed on it, and the circular sponge support of Α in Figure 8 is prepared according to the photocrosslinking method described in Example 1 The inner diameter of the ring is 2400μηι, the outer diameter is 3400μηι _; the height of the sponge stent is 300μηι _; the pore size of the sponge stent is 10μηι-125μηΐ; the pore spacing is 1μηι-999μηι; the porosity is 93.3%, the connectivity is good; It is 3.5-5 times of the theoretical volume; the sponge scaffold array consists of 24 sponge scaffolds.) Covered in a thin layer of cell liquid (thick layer thickness of 50 μm), after the cell solution is dispersed into the inside of the sponge scaffold, light Lightly remove the scaffold array slides to achieve high-throughput three-dimensional loading of HeLa cells (Figure 8, A, HeLa cells: green-Calcein-AM). The effect of the liquid layer loading of different cell concentrations is shown in Figure 8 B-1 to B-3 (B-1: 2X 10 ⁶ /ml, B-2: 4X 10 ⁵ /ml, B-3: 8 X 10 ⁴ / ml). Confocal laser scanning microscope cell distribution within Sponges, see FIG. 8 C-1 to C-3 (Cl: 2X 10 6 th / ml, C-2: 4X 10 5 th / ml, C-3: 8 X 10 ⁴ / ml). The results showed that as the cell concentration increased, the more cells were automatically loaded into the sponge scaffold, the stronger the green fluorescence intensity, and the denser the three-dimensional distribution of cells, especially B-1 and Cl in Fig. 8. This demonstrates that the above-described high-throughput sponge scaffold display enables automatic loading of HeLa cells by the liquid thin layer method.

接着，采用 Cell Titer-Blue试剂定量液体薄层法自动装载细胞的效率。 Next, Cell Titer-Blue reagent was used to quantify the efficiency of the liquid thin layer method for autoloading cells.

1、建立活细胞数量-荧光强度标准曲线； 1. Establish a standard curve of the number of living cells - fluorescence intensity;

在 96孔板中分别种植 4X 10⁴个 /孔、 8 X 10³个 /孔、 1.6 X 10³个 /孔、 320个 /孔的一系列浓度的 HeLa细胞，每个浓度重复 3个样本，每个孔中加入 ΙΟΟμΙ培养液和 20μ1的 Cell Titer-Blue试剂，在 37°C、 5 C0₂ 饱和湿度培养箱中孵育 lh。用酶标仪测定荧光强度，建立标准曲线（图 8中 E) 。 A series of concentrations of HeLa cells were implanted in a 96-well plate at ⁴ ×10 ⁴ /well, 8×10 ³ /well, 1.6×10 ³ /well, 320/well, and 3 samples were repeated for each concentration. ΙΟΟμΙ culture solution and 20 μl of Cell Titer-Blue reagent were added to each well, and incubated at 37 ° C in a 5 C0 ₂ saturated humidity incubator for 1 h. The fluorescence intensity was measured with a microplate reader to establish a standard curve (E in Figure 8).

2、 Cell Titer-Blue试剂定量液体薄层法自动装载细胞的效率； 2. Cell Titer-Blue reagent quantitative liquid layer method for automatic loading of cells;

按上述液体薄层法分别将不同细胞浓度的悬浮液自动装载到高通量海绵支架阵列 (基底 B及固定其上的海绵支架阵列，图 8中 A所示的圆环形海绵支架）中。在每个液体薄层载玻片（基底 A)和海绵支架载玻片（基底 B及固定其上的海绵支架阵列）上分别加入 ΙΟΟΟμΙ培养液和 200μ1的 Cell Titer-Blue试剂混合液，在 37°C、 5 C0₂ 饱和湿度培养箱中孵育 lh (图 8， D-l : 5 X 10⁶个 /ml， D-2: 1 X 10⁶个 /ml, D-3: 2X 10⁵个 /ml)。从每个混合液中取 30μ1到 384孔板，用酶标仪测定荧光强度，根据标准曲线（图 8中 Ε)，定量液体薄层法自动高通量装载不同浓度 HeLa细胞进入海绵支架的效率，见图 8中 F。结果表明，随着细胞浓度的提高，海绵支架内的细胞与 Cell Titer-Blue试剂反应，导致其颜色越偏红，说明自动装载进入海绵支架的细胞越多，液体薄层法实现自动装载的效率越高。 Suspensions of different cell concentrations were automatically loaded into the high-throughput sponge scaffold array (substrate B and the sponge scaffold array immobilized thereon, in the circular sponge scaffold shown in A of Fig. 8) according to the above liquid thin layer method. On each of the liquid thin slides (substrate A) and the sponge scaffold slides (substrate B and the sponge scaffold array immobilized thereon), a mixture of ΙΟΟΟμΙ culture solution and 200 μl of Cell Titer-Blue reagent was separately added, at 37 Incubate for 1 h in °C, 5 C0 ₂ saturated humidity incubator (Fig. 8, Dl: 5 X 10 ⁶ /ml, D-2: 1 X 10 ⁶ /ml, D-3: 2X 10 ⁵ /ml) . Take 30μ1 to 384-well plate from each mixture, measure the fluorescence intensity with a microplate reader, and calculate the efficiency of automatic high-throughput loading of HeLa cells into the sponge scaffold by quantitative liquid layer method according to the standard curve (Ε in Figure 8). , see Figure F, F. The results showed that as the cell concentration increased, the cells in the sponge scaffold reacted with the Cell Titer-Blue reagent, resulting in a reddish color, indicating that the more cells were automatically loaded into the sponge scaffold, the efficiency of the liquid thin layer method for automatic loading. The higher.

工业应用 Industrial application

本发明提出了一种基于透明海绵支架材料实现能够和两维细胞培养方法一样简易，方便操作（easy as 2D) 的构建三维细胞微环境的方法，并同时满足图案化、高通量、实时无标记监测等研究目的。 1 ) 利用三维海绵支架材料的多孔性实现了自动简易的细胞或细胞-材料的装载。采用传统滴加方法，细胞或细胞-材料悬浮液便能自动吸附进入多孔海绵支架内部，形成三维微环境体系； 2) 种植细胞在三维微环境中可是实现自由三维生长，增殖，并且易于传代； 3 ) 利用透明海绵支架材料的优良光学性能，实现应用常规设备（如普通光学显微镜）对细胞进行无标记观测； 4) 透明海绵支架材料可有效结合三维微加工技术，实现三维微环境的微量化、图案化以形成高通量三维微环境阵列； 5 ) 我们还发明了一种简易、可广泛应用的液体薄层法，可以快速、无损地实现分子、材料和细胞以及其混合物的三维微环境的高通量同步装载。 The invention proposes a method for constructing a three-dimensional cell microenvironment based on the transparent sponge scaffold material, which is as simple and convenient as the two-dimensional cell culture method, and satisfies the patterning, high-throughput, real-time without Marking monitoring and other research purposes. 1) The automatic and simple loading of cells or cells-materials is achieved by the porosity of the three-dimensional sponge scaffold material. With the traditional dropping method, the cell or cell-material suspension can be automatically adsorbed into the porous sponge scaffold to form a three-dimensional microenvironment system; 2) the planting cells can realize free three-dimensional growth, proliferation, and easy passage in the three-dimensional microenvironment; 3) Using the excellent optical properties of the transparent sponge scaffold material, the conventional equipment (such as ordinary optical microscope) can be used to perform unmarked observation on the cells; 4) The transparent sponge scaffold material can effectively combine the three-dimensional micro-machining technology to realize the micro-environment of the three-dimensional micro-environment , patterned to form a high-throughput three-dimensional microenvironment array; 5) We have also invented a simple, widely applicable liquid thin layer method that enables rapid, non-destructive realization of the three-dimensional microenvironment of molecules, materials and cells, and mixtures thereof. High-throughput synchronous loading.

本发明与现有研究相比具有如下优点： 1、本发明方法结合工程学、化学、物理学、材料学等学科知识，制备三维透明海绵多孔支架，利用支架的多孔性、光学透明性、机械弹性、交联可控性等特征，为生物学、药学、医学等领域对于精确可控和高通量的三维细胞微环境的研究提供一个简单易行、应用广泛的平台； 2、本发明提出的三维微环境构建方法与传统两维细胞培养手段一样简单易行（easy as 2D ) ，使用常规设备 ( off-the-shelf instruments )满足细胞在三维体系中快速种植、自由生长、长期增殖、易于传代、实时监测等基本培养要求； 3、本发明提出的透明海绵支架概念填补了材料性能领域的空白。本发明提供的制备透明海绵支架的方法有效地改善了传统三维支架光学性能差的问题，可满足生物学、药学、医学等领域对三维微环境进行实时无标记成像研究的要求，极大地降低了对研究手段、方法、设备等方面的要求； 4、本发明提出的液体薄层装载方法，无需特别的专业技术和手段以及昂贵的设备就可以实现三维细胞微环境的高通量同步构建，大大降低了操作过程中对于人员技能、环境空间等各方面的使用要求，具有广泛的应用前景，且简易的操作步骤降低了对活细胞的损伤； 5、本发明方法可与各种现有研究技术（如三维微加工技术、细胞动态种植技术、自动化操作***等）结合使用，以实现图案化（patterning) 、微观结构（microstructure) 可控性，实时监测 (monitoring)等目的，来满足不同研究领域、不同研究目的的多重需求； 6、本发明最终可应用于开发各种三维细胞应用产品，为三维细胞微环境研究提供现成的 ( off-the-shelf)平台，以实现三维细胞产品在传统生物学、药学和医学领域的无障碍广泛应用。 Compared with the prior research, the invention has the following advantages: 1. The method of the invention combines engineering, chemistry, physics, material science and other subject knowledge to prepare a three-dimensional transparent sponge porous stent, utilizing the porosity, optical transparency, mechanical of the stent Elasticity, cross-linking controllability, etc., provide a simple and widely applicable platform for the study of precise and controllable and high-throughput three-dimensional cellular microenvironment in the fields of biology, pharmacy, medicine, etc. 2. The present invention proposes The three-dimensional microenvironment construction method is as simple as the traditional two-dimensional cell culture method (easy as 2D), using the off-the-shelf instruments to satisfy the rapid planting, free growth, long-term proliferation, and easy of cells in a three-dimensional system. Basic culture requirements such as passage and real-time monitoring; 3. The transparent sponge stent concept proposed by the present invention fills a gap in the field of material properties. The method for preparing a transparent sponge stent provided by the invention effectively improves the problem of poor optical performance of the traditional three-dimensional stent, and can meet the requirements of real-time unmarked imaging research on the three-dimensional micro-environment in the fields of biology, pharmacy, medicine, etc., and greatly reduces the requirement. Requirements for research methods, methods, equipment, etc. 4. The liquid thin layer loading method proposed by the present invention can realize high-throughput synchronous construction of a three-dimensional cellular micro-environment without special expertise and means and expensive equipment, It reduces the use requirements of personnel skills, environmental space and other aspects during operation, has broad application prospects, and simple operation steps reduce damage to living cells; 5. The method of the present invention can be combined with various existing research techniques (such as three-dimensional micro-machining technology, cell dynamic planting technology, automated operating system, etc.) combined to achieve patterning, microstructure controllability, real-time monitoring (monitoring) and other purposes to meet different research areas Multiple needs for different research purposes 6. The present invention can be finally applied to the development of various three-dimensional cell application products, providing an off-the-shelf platform for three-dimensional cellular microenvironment research to realize the three-dimensional cellular products in the fields of traditional biology, pharmacy and medicine. Barriers are widely used.

此发明方法对于熟悉传统两维细胞培养、研究的人员来说操作简单，无需其他的专业技术和手段（如微加工技术和特殊合成材料）以及昂贵的设备（如自动化、微加工设备）。最终以实现在传统生物学、药学和医学领域的无障碍广泛应用。 The method of the invention is simple to operate for those familiar with conventional two-dimensional cell culture and research, without the need for other specialized techniques and means (such as micromachining techniques and special synthetic materials) and expensive equipment (such as automation, micromachining equipment). Ultimately, it is widely used in the field of traditional biology, pharmacy and medicine.

Claims

权利要求 Rights request

1、用于构建三维微环境的海绵支架，其特征在于：所述海绵支架为透明的海绵支架，所述透明的海绵支架为透明度达到 50%以上的所述海绵支架； A sponge stent for constructing a three-dimensional microenvironment, wherein: the sponge stent is a transparent sponge stent, and the transparent sponge stent is a sponge stent having a transparency of 50% or more;

所述海绵支架用生物材料制成，具有若干小孔；所述小孔的孔径为 1ηηι-999μηι，孔间距为 1μηι-999μηι，所述小孔在所述海绵支架上所形成的孔隙率为 70%-99.9%；所述海绵支架的体积为 0.^m³-1000cm³; The sponge stent is made of a biological material and has a plurality of small holes; the pores have a pore diameter of 1ηηι-999μηι, a pore spacing of 1μηι-999μηι, and a porosity of the small pores formed on the sponge stent is 70 %-99.9%; the volume of the sponge scaffold is 0. ^ m ³ -1000cm ³ ;

2、用于构建三维微环境的装置，包括下述 A1 ) 和 A2) 的两个器件： A1 ) 权利要求 1所述海绵支架或由两个以上所述海绵支架组成的海绵支架阵列； A2)用于装载样品液的基底 A; 2. Apparatus for constructing a three-dimensional microenvironment comprising two devices of the following A1) and A2): A1) a sponge scaffold according to claim 1 or an array of sponge scaffolds composed of two or more sponge scaffolds; A2) a substrate A for loading a sample liquid;

所述装载样品液的基底 A为亲水性基底或疏水性基底；所述基底的亲水性或疏水性使样品液在所述基底的表面形成液体薄层；所述液体薄层的厚度为 1μηι -200μηι。 The substrate A carrying the sample liquid is a hydrophilic substrate or a hydrophobic substrate; the hydrophilicity or hydrophobicity of the substrate causes the sample liquid to form a thin liquid layer on the surface of the substrate; the thickness of the liquid thin layer is 1μηι -200μηι.

3、根据权利要求 1所述的海绵支架，或权利要求 2所述的装置，其特征在于：在制备所述透明的海绵支架的过程中，使用透明剂或 /和微加工。 3. A sponge scaffold according to claim 1, or a device according to claim 2, characterized in that a transparent agent or/and micromachining is used in the preparation of the transparent sponge scaffold.

4、根据权利要求 3所述的海绵支架或所述的装置，其特征在于：通过所述微加工实现所述海绵支架的图案化，和 /或实现所述海绵支架阵列的高通量。 4. Sponge holder or device according to claim 3, characterized in that the patterning of the sponge holder is achieved by the micromachining and/or the high throughput of the sponge holder array is achieved.

5、根据权利要求 4所述的海绵支架或所述的装置，其特征在于：所述微加工为通过光掩膜的透光部分的图形实现所述海绵支架的图案化，和 /或实现所述海绵支架阵列的高通量。 5. The sponge holder or device of claim 4, wherein: said micromachining is to effect patterning of said sponge holder through a pattern of light transmissive portions of the photomask, and/or to achieve High throughput of the sponge scaffold array.

6、根据权利要求 2-4中任一所述的装置，其特征在于：所述海绵支架阵列由 3个以上的所述海绵支架组成，形成高通量的海绵支架阵列。 6. Apparatus according to any of claims 2-4, wherein: said sponge scaffold array consists of more than three of said sponge scaffolds to form a high throughput sponge scaffold array.

7、根据权利要求 3所述的海绵支架或所述的装置，其特征在于：所述透明的海绵支架按照包括如下步骤的方法制备： 7. A sponge scaffold or apparatus according to claim 3, wherein: said transparent sponge scaffold is prepared in accordance with the method comprising the steps of:

bl )将聚合物单体聚乙二醇二丙烯酸酯和光引发剂 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮溶解在所述透明剂与水的混合溶液中，得到可光交联的预聚物溶液 A; Bl) dissolving the polymer monomer polyethylene glycol diacrylate and the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in a mixed solution of the transparent agent and water , obtaining a photocrosslinkable prepolymer solution A;

b3)将步骤 b2)获得的所述水凝胶浸于超纯水中除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质。 B3) The hydrogel obtained in the step b2) is immersed in ultrapure water to remove the uncrosslinked polymer monomer polyethylene glycol diacrylate, the clearing agent and impurities.

8、根据权利要求 7所述的海绵支架或所述的装置，其特征在于：所述聚合物单体聚乙二醇二丙烯酸酯在所述可光交联的预聚物溶液 A中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 l-50g所述聚合物单体聚乙二醇二丙烯酸酯； 8. The sponge scaffold or the device according to claim 7, wherein: the content of the polymer monomer polyethylene glycol diacrylate in the photocrosslinkable prepolymer solution A For each 100 ml of the photocrosslinkable The prepolymer solution A contains 1-50 g of the polymer monomer polyethylene glycol diacrylate;

所述光引发剂 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮在所述可光交联的预聚物溶液 A 中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 0.1-10g所述 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮。 The content of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the photocrosslinkable prepolymer solution A is 100 mg per liter of the photocrossable The prepolymer solution A contained 0.1 to 10 g of the 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

9、根据权利要求 7所述的海绵支架或所述的装置，其特征在于：所述透明剂在所述透明剂与水的混合溶液中的体积百分含量大于等于 0.01%，同时小于 100%。 9. The sponge holder or the device according to claim 7, wherein: the transparent agent has a volume percentage of the transparent agent and water mixed solution of 0.01% or more and less than 100%. .

10、根据权利要求 7所述的海绵支架或所述的装置，其特征在于:所述透明剂为 1,2,4- 丁三醇、乙二醇， 1,3-丁二醇，丙三醇， 1,2-丙二醇， 1,3-丙二醇，季戊四醇，顺 -1,2-环戊二醇，丁四醇和戊五醇等多元醇中的至少一种。 10. A sponge scaffold or apparatus according to claim 7, wherein said clearing agent is 1,2,4-butanetriol, ethylene glycol, 1,3-butanediol, and C3 At least one of an alcohol such as 1,2-propanediol, 1,3-propanediol, pentaerythritol, cis-1,2-cyclopentanediol, butanol and pentaerythritol.

11、根据权利要求 7所述的海绵支架或所述的装置，其特征在于：所述聚合物单体聚乙二醇二丙烯酸酯在所述可光交联的预聚物溶液 A中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 10g所述聚合物单体聚乙二醇二丙烯酸酯； The sponge holder or the device according to claim 7, wherein: the content of the polymer monomer polyethylene glycol diacrylate in the photocrosslinkable prepolymer solution A 10 g of the polymer monomer polyethylene glycol diacrylate per 100 ml of the photocrosslinkable prepolymer solution A;

所述光引发剂 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮在所述可光交联的预聚物溶液 A 中的含量为每 100ml所述可光交联的预聚物溶液 A中含有 0.5g所述 2-羟基 -4-(2-羟乙氧基) -2-甲基苯丙酮； The content of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in the photocrosslinkable prepolymer solution A is 100 mg per liter of the photocrossable The prepolymer solution A contains 0.5 g of the 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone;

所述透明剂具体为 1,2,4-丁三醇，所述 1,2,4-丁三醇在所述透明剂与水的混合溶液中的体积百分含量具体为 60%。 The clearing agent is specifically 1,2,4-butanetriol, and the volume percentage of the 1,2,4-butanetriol in the mixed solution of the clearing agent and water is specifically 60%.

12、根据权利要求 3所述的海绵支架或所述的装置，其特征在于：所述透明的海绵支架按照包括如下步骤的方法制备： 12. A sponge scaffold or apparatus according to claim 3, wherein: said transparent sponge scaffold is prepared in accordance with the method comprising the steps of:

c2) 在步骤 cl ) 得到预聚物溶液 B后，将其加到制备海绵支架的模具中，使所述预聚物溶液 B发生化学交联反应，得到水凝胶； C2) after obtaining the prepolymer solution B in step cl), adding it to a mold for preparing a sponge scaffold, and chemically crosslinking the prepolymer solution B to obtain a hydrogel;

13、根据权利要求 12所述的海绵支架或所述的装置，其特征在于：所述聚合物单体聚乙二醇二丙烯酸酯在所述预聚物溶液 B中的含量为每 100ml所述预聚物溶液 B中含有 l-50g所述聚合物单体聚乙二醇二丙烯酸酯； The sponge holder or the device according to claim 12, wherein: the content of the polymer monomer polyethylene glycol diacrylate in the prepolymer solution B is 100 mg per 100 ml. The prepolymer solution B contains 1-50 g of the polymer monomer polyethylene glycol diacrylate;

所述过硫酸铵在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 0.01-lg所述过硫酸铵； The ammonium persulfate may be contained in the prepolymer solution B in an amount of 0.01-lg of the ammonium persulfate per 100 ml of the prepolymer solution B;

所述 Ν,Ν,Ν',Ν'-四甲基二乙胺在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 0.01-lg所述 Ν,Ν,Ν',Ν'-四甲基二乙胺。 The content of the ruthenium, osmium, iridium, Ν'-tetramethyldiethylamine in the prepolymer solution B may be 0.01-lg per 100 ml of the prepolymer solution B. ,Ν',Ν'-tetramethyldiethylamine.

14、根据权利要求 12所述的海绵支架或所述的装置，其特征在于：所述透明剂在所述透明剂与水的混合溶液中的体积百分含量大于等于 0.01%，同时小于 100%。 The sponge holder or the device according to claim 12, wherein: the transparent agent has a volume percentage of the transparent agent and water in a mixed solution of 0.01% or more and less than 100%. .

15、根据权利要求 12所述的海绵支架或所述的装置，其特征在于：所述透明剂为 The sponge holder or the device according to claim 12, wherein: the transparent agent is

1,2,4-丁三醇、乙二醇， 1,3-丁二醇，丙三醇， 1,2-丙二醇， 1,3-丙二醇，季戊四醇，顺 -1,2- 环戊二醇，丁四醇和戊五醇等多元醇中的至少一种。 1,2,4-butanetriol, ethylene glycol, 1,3-butanediol, glycerol, 1,2-propanediol, 1,3-propanediol, pentaerythritol, cis-1,2-cyclopentanediol At least one of a polyhydric alcohol such as tetramethylene alcohol and pentaerythritol.

16、根据权利要求 12所述的海绵支架或所述的装置，其特征在于：所述聚合物单体聚乙二醇二丙烯酸酯在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B 中含有 10g所述聚合物单体聚乙二醇二丙烯酸酯； 16. The sponge holder or device of claim 12, wherein: said polymer sheet The content of the polyethylene glycol diacrylate in the prepolymer solution B may be 10 g of the polymer monomer polyethylene glycol diacrylate per 100 ml of the prepolymer solution B;

所述过硫酸铵在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 0.05g所述过硫酸铵； The ammonium persulfate may be contained in the prepolymer solution B in an amount of 0.05 g of the ammonium persulfate per 100 ml of the prepolymer solution B;

所述 Ν,Ν,Ν',Ν'-四甲基二乙胺在所述预聚物溶液 B中的含量可为每 100ml所述预聚物溶液 B中含有 0.5g所述 Ν,Ν,Ν',Ν'-四甲基二乙胺； The content of the hydrazine, hydrazine, hydrazine, Ν'-tetramethyldiethylamine in the prepolymer solution B may be 0.5 g of the strontium, strontium per 100 ml of the prepolymer solution B. Ν', Ν'-tetramethyldiethylamine;

所述透明剂具体为 1,2,4-丁三醇，所述 1,2,4-丁三醇在所述透明剂与水的混合溶液中的体积百分含量为 60%。 The clearing agent is specifically 1,2,4-butanetriol, and the volume percentage of the 1,2,4-butanetriol in the mixed solution of the clearing agent and water is 60%.

17、根据权利要求 7或 12所述的海绵支架或所述的装置，其特征在于：在除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质后，还包括如下步骤：将所述水凝胶在 -200°C ~0°C条件下冷冻 l-72h，再干燥 1-72小时，得到所述海绵支架或所述海绵支架阵列。 The sponge holder or the device according to claim 7 or 12, wherein: after removing the uncrosslinked polymer monomer polyethylene glycol diacrylate, the transparent agent and impurities The method further comprises the steps of: freezing the hydrogel at -200 ° C to 0 ° C for 1-72 h, and drying for 1-72 hours to obtain the sponge scaffold or the sponge scaffold array.

18、根据权利要求 17所述的海绵支架或所述的装置，其特征在于：在除去未交联的所述聚合物单体聚乙二醇二丙烯酸酯、所述透明剂和杂质后，还包括如下步骤：将所述水凝胶在 -20°C条件下冷冻 4-5h，再在 -50 °C， 20pa条件下，干燥 12小时，得到所述海绵支架或所述海绵支架阵列。 The sponge holder or the device according to claim 17, wherein: after removing the uncrosslinked polymer monomer polyethylene glycol diacrylate, the transparent agent and impurities, The method includes the following steps: freezing the hydrogel at -20 ° C for 4-5 h, and drying at -50 ° C, 20 Pa for 12 hours to obtain the sponge scaffold or the sponge scaffold array.

19、根据权利要求 12所述的海绵支架或所述的装置，其特征在于：所述制备海绵支架的模具用生物材料制成，所述生物材料为人工合成的生物材料和 /或可交联天然生物材料；所述人工合成的生物材料为下述至少一种：聚甲基丙烯酸酯、聚乙二醇、聚乙二醇衍生物、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、聚縮醛、聚氰基丙烯酸酯、聚氨基甲酸酯、聚吡咯、聚酯、聚乙烯、聚碳酸酯和聚氧化乙烯；所述天然生物材料为下述至少一种：明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、蛋白多糖、糖蛋白、透明质酸、层连接蛋白和纤维连接蛋白。 19. A sponge scaffold or device according to claim 12, wherein: said mold for preparing a sponge scaffold is made of a biomaterial which is a synthetic biomaterial and/or crosslinkable a natural biomaterial; the synthetic biomaterial is at least one of the following: polymethacrylate, polyethylene glycol, polyethylene glycol derivative, polypropylene, polystyrene, polyacrylamide, polylactic acid, Polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole , polyester, polyethylene, polycarbonate, and polyethylene oxide; the natural biomaterial is at least one of the following: gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, Proteoglycans, glycoproteins, hyaluronic acid, laminin and fibronectin.

20、根据权利要求 2所述的装置，其特征在于：所述装置还包括镶嵌在所述基底 A 上的边框； 20. The device according to claim 2, wherein: the device further comprises a frame embedded on the substrate A;

21、根据权利要求 2所述的装置，其特征在于：所述基底 A为亲水性基底；所述基底 A上的边框用聚甲基丙烯酸甲酯制成。 The apparatus according to claim 2, wherein: said substrate A is a hydrophilic substrate; and the frame on said substrate A is made of polymethyl methacrylate.

22、根据权利要求 2所述的装置，其特征在于：所述装置还包括支撑所述海绵支架或所述海绵支架阵列的另一基底 B。 22. Apparatus according to claim 2 wherein: said apparatus further comprises another substrate B supporting said sponge scaffold or said array of sponge scaffolds.

23、根据权利要求 1所述的海绵支架，或权利要求 2所述的装置，其特征在于：所述三维微环境为包括下述 a) -d) 中任一所述的三维微环境： 23. A sponge support according to claim 1 or a device according to claim 2, wherein: The three-dimensional microenvironment is a three-dimensional microenvironment comprising any one of the following a) -d):

a) 小分子化合物、药物、核酸、蛋白中的任一种或任几种的混合物； a) a mixture of any one or a combination of a small molecule compound, a drug, a nucleic acid, a protein;

b) 细胞外基质成分、高分子材料、微珠中的任一种或任几种的混合物； b) any one or a mixture of extracellular matrix components, polymeric materials, microbeads;

c) 真核细胞、原核细胞、病毒、微生物中的任一种或任几种的混合物； c) any one or a mixture of any one of eukaryotic cells, prokaryotic cells, viruses, microorganisms;

d) a) -c) 中任几种的混合物。 d) a mixture of any of a) -c).

24、权利要求 1所述的海绵支架，或权利要求 2所述的装置在构建三维微环境中的应用。 24. Use of the sponge scaffold of claim 1 or the device of claim 2 in constructing a three dimensional microenvironment.

25、根据权利要求 24所述的应用，其特征在于：所述三维微环境为包括下述 a) -d) 中任一所述的三维微环境： The application according to claim 24, wherein: said three-dimensional microenvironment is a three-dimensional microenvironment comprising any one of the following a) - d):

d) a) -c) 中任几种的混合物。 d) a mixture of any of a) -c).

26、构建三维微环境的方法为用权利要求 2所述装置构建三维微环境，包括如下步骤： 26. A method of constructing a three-dimensional microenvironment for constructing a three-dimensional microenvironment using the apparatus of claim 2, comprising the steps of:

al ) 将样品液置于权利要求 2所述装置的基底 A上，形成液体薄层； Al) placing the sample liquid on the substrate A of the apparatus of claim 2 to form a thin layer of liquid;

a2) 将步骤 al ) 中形成所述液体薄层的所述基底 A覆盖在权利要求 2所述装置的海绵支架或海绵支架阵列上，或者将权利要求 2所述海绵支架或所述海绵支架阵列覆盖在步骤 al ) 中形成所述液体薄层的所述基底 A上，待所述样品液分散进入到所述海绵支架或所述海绵支架阵列后，实现所述样品液的三维尺度装载，完成所述三维微环境的构建； A2) covering the substrate A forming the thin layer of the liquid in the step a1 on the sponge or sponge scaffold array of the device of claim 2, or the sponge scaffold or the sponge scaffold array according to claim 2. Covering the substrate A forming the thin layer of the liquid in the step a1, after the sample liquid is dispersed into the sponge stent or the sponge stent array, the three-dimensional loading of the sample liquid is completed, and the completion is completed. Construction of the three-dimensional microenvironment;

所述液体薄层的厚度为 1μηι -200μηι。 The thin layer of liquid has a thickness of 1 μηι - 200 μηι.

27、根据权利要求 26所述的方法，其特征在于：所述样品液包括下述 a) -d) 中任一所述的物质： 27. The method according to claim 26, wherein: the sample liquid comprises the substance according to any one of the following a) - d):

d) a) -c) 中任几种的混合物。 d) a mixture of any of a) -c).