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CN117565386A - A cell or organoid chip and its preparation method and application - Google Patents

A cell or organoid chip and its preparation method and application Download PDF

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Publication number
CN117565386A
CN117565386A CN202410067558.5A CN202410067558A CN117565386A CN 117565386 A CN117565386 A CN 117565386A CN 202410067558 A CN202410067558 A CN 202410067558A CN 117565386 A CN117565386 A CN 117565386A
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cell
hydrogel
substrate
precursor
organoid
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CN117565386B (en
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苏萌
谢岱希
陈炳达
宋延林
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Institute of Chemistry CAS
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/307—Handling of material to be used in additive manufacturing
    • B29C64/314—Preparation
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    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
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    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B33Y40/10—Pre-treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
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Abstract

本发明提供了一种细胞或类器官芯片及其制备方法与应用，该方法包括以下步骤：S1：制备生物活性墨水，所述生物活性墨水包括分散介质和生物单元；S2：在基底表面制造微槽，得到预处理的基底；S3：将预处理的基底进行表面修饰，得到表面修饰的基底；S4：将所述生物活性墨水经3D打印方法打印到表面修饰的基底上，得到所述细胞或类器官芯片。本发明通过打印技术制备具有高粘附性的药物筛选芯片，可以利用少量样本实现药物筛选。

The invention provides a cell or organoid chip and its preparation method and application. The method includes the following steps: S1: prepare bioactive ink, the bioactive ink includes a dispersion medium and a biological unit; S2: manufacture microorganisms on the surface of a substrate. groove to obtain a pre-treated substrate; S3: Surface-modify the pre-treated substrate to obtain a surface-modified substrate; S4: Print the bioactive ink onto the surface-modified substrate through 3D printing method to obtain the cells or Organoids on a chip. The present invention prepares a drug screening chip with high adhesion through printing technology, and can realize drug screening using a small amount of samples.

Description

一种细胞或类器官芯片及其制备方法与应用A cell or organoid chip and its preparation method and application

技术领域Technical field

本发明涉及细胞或组织培养技术领域，尤其涉及一种细胞或类器官芯片及其制备方法与应用。The present invention relates to the technical field of cell or tissue culture, and in particular to a cell or organoid chip and its preparation method and application.

背景技术Background technique

随着3D打印技术的不断发展、应用和推广，使用和体验的人越来越多，应用的领域也是越来越广泛了。如何将3D打印应用于细胞培养也已成为又一个热点，例如，骨折、骨损、骨肿瘤等骨科医疗科学的骨细胞研究，皮组织细胞、口腔细胞、内脏各器官细胞等的培养，越来越多的科研人员都在研究，也报道或上市了一些产品。With the continuous development, application and promotion of 3D printing technology, more and more people are using and experiencing it, and the fields of application are becoming more and more extensive. How to apply 3D printing to cell culture has also become another hot spot. For example, research on bone cells in orthopedic medical sciences such as fractures, bone damage, and bone tumors, and the culture of skin tissue cells, oral cells, and visceral organ cells, etc., are becoming more and more popular. More and more scientific researchers are doing research, and some products have been reported or launched.

传统的细胞培养装置、设备或支架都属于二维的平面培养模式，虽然在一定程度上可以满足很大一部分的产业需求，但也存在一些不足或缺陷，比如细胞在培养过程中增殖后细胞出现拥挤，部分无黏附着床空间，暴露在培养液表面等。三维培养支架的出现很好地解决了二维培养所存在的不足与缺陷，三维支架丰富的网络空间结构可以给细胞的增殖和黏附提供更大的附着比表面积，提高细胞的产量和质量，减少细胞之间彼此的接触和抑制，具有模拟人体或动物体的三维结构生长环境，有利于细胞与细胞之间及细胞与细胞外质之间的交互作用及代谢产物的排除，有利于细胞的分化及原属性功能的表达。Traditional cell culture devices, equipment or scaffolds are all two-dimensional planar culture models. Although they can meet a large part of industrial needs to a certain extent, they also have some shortcomings or defects, such as the appearance of cells after cell proliferation during the culture process. Crowded, partially non-adherent bed space, exposed to culture medium surface, etc. The emergence of three-dimensional culture scaffolds has well solved the shortcomings and defects of two-dimensional culture. The rich network space structure of three-dimensional scaffolds can provide a larger specific surface area for cell proliferation and adhesion, improve cell yield and quality, and reduce The contact and inhibition between cells create a three-dimensional structural growth environment that simulates the human or animal body, which is conducive to the interaction between cells and cells and the extracellular substance of cells and the elimination of metabolic products, and is conducive to cell differentiation. and the expression of original attribute functions.

目前大部分的三维培养支架以聚己内酯等为原料，采用凝胶点胶或打印成型等得到三维细胞培养支架。由于其疏水性，不利于细胞的附着贴壁。故此，亟待开发一种具有良好的细胞亲和性、粘附性的三维细胞培养支架来满足生产发展的需求。At present, most of the three-dimensional culture scaffolds are made of polycaprolactone and other raw materials, and the three-dimensional cell culture scaffolds are obtained by gel dispensing or printing. Due to its hydrophobicity, it is not conducive to cell adhesion. Therefore, there is an urgent need to develop a three-dimensional cell culture scaffold with good cell affinity and adhesion to meet the needs of production development.

发明内容Contents of the invention

针对现有技术中所存在的不足，本发明提供了一种细胞或类器官芯片及其制备方法和应用，其解决了现有技术中存在的细胞或类器官芯片粘附性差的问题。In view of the deficiencies in the prior art, the present invention provides a cell or organoid chip and its preparation method and application, which solves the problem of poor adhesion of the cell or organoid chip existing in the prior art.

本发明一方面提供了一种细胞或类器官芯片，具体的是一种高粘附性细胞或类器官芯片，其表面存在微槽、以及高粘附性分子层。本发明另一方面提供了一种细胞或类器官芯片的制备方法，具体的是一种高粘附性细胞或类器官芯片的制备方法。In one aspect, the present invention provides a cell or organoid chip, specifically a highly adhesive cell or organoid chip with microgrooves and a highly adhesive molecular layer on its surface. Another aspect of the present invention provides a method for preparing a cell or organoid chip, specifically a method for preparing a highly adhesive cell or organoid chip.

具体的，本发明提供如下技术方案：Specifically, the present invention provides the following technical solutions:

一种细胞或类器官芯片的制备方法，具体的是一种高粘附性细胞或类器官芯片的制备方法，所述制备方法包括以下步骤：A method for preparing a cell or organoid chip, specifically a method for preparing a highly adhesive cell or organoid chip. The preparation method includes the following steps:

S1：制备生物活性墨水，所述生物活性墨水包括分散介质和生物单元；S1: Preparing a bioactive ink, which includes a dispersion medium and a biological unit;

S2：在基底表面制造微槽，得到预处理的基底；S2: Create microgrooves on the surface of the substrate to obtain a pretreated substrate;

S3：将预处理的基底进行表面修饰，得到表面修饰的基底；S3: Surface-modify the pre-treated substrate to obtain a surface-modified substrate;

S4：将所述生物活性墨水经3D打印方法打印到表面修饰的基底上，得到所述细胞或类器官芯片。S4: Print the bioactive ink onto the surface-modified substrate through 3D printing to obtain the cell or organoid chip.

在本发明一实施方式中，所述分散介质至少包括水凝胶交联前体和水凝胶粘附增强剂。In one embodiment of the present invention, the dispersion medium at least includes a hydrogel cross-linking precursor and a hydrogel adhesion enhancer.

在本发明一实施方式中，所述分散介质中，水凝胶交联前体和水凝胶粘附增强剂的质量比为(1~10):0.5；示例性地，为1:0.5、2:0.5、3:0.5、4:0.5、5:0.5、6:0.5、7:0.5、8:0.5、9:0.5、10:0.5或介于其中的某一具体比例值。In one embodiment of the present invention, in the dispersion medium, the mass ratio of the hydrogel cross-linking precursor and the hydrogel adhesion enhancer is (1~10):0.5; exemplarily, it is 1:0.5, 2:0.5, 3:0.5, 4:0.5, 5:0.5, 6:0.5, 7:0.5, 8:0.5, 9:0.5, 10:0.5 or a specific ratio value between them.

在本发明一实施方式中，所述分散介质至少包括水凝胶交联前体，任选添加或不添加交联引发剂。当添加交联引发剂时，在形成生物墨水时所述水凝胶交联前体能够与对应的交联引发剂作用形成水凝胶。In one embodiment of the present invention, the dispersion medium at least includes a hydrogel cross-linking precursor, optionally with or without the addition of a cross-linking initiator. When a cross-linking initiator is added, the hydrogel cross-linking precursor can react with the corresponding cross-linking initiator to form a hydrogel when forming the bioink.

在本发明一实施方式中，所述交联引发剂选自光引发剂、离子引发剂中的一种或几种。In one embodiment of the present invention, the cross-linking initiator is selected from one or more types of photoinitiators and ionic initiators.

在本发明一实施方式中，所述光引发剂包括但不限于苯基-2,4,6-三甲基苯甲酰基亚磷酸锂(简称LAP)、2-羟基-4′-(2-羟乙氧基)-2-甲基苯丙酮（I2959）、2,4,6-三甲基苯甲酰基苯基膦酸乙酯（TPO-L）中的至少一种。In one embodiment of the present invention, the photoinitiator includes but is not limited to phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP for short), 2-hydroxy-4'-(2- At least one of hydroxyethoxy)-2-methylpropiophenone (I2959) and ethyl 2,4,6-trimethylbenzoylphenylphosphonate (TPO-L).

在本发明一实施方式中，所述离子引发剂包括但不限于氯化钙溶液、氯化钡溶液中的至少一种。In one embodiment of the present invention, the ion initiator includes but is not limited to at least one of calcium chloride solution and barium chloride solution.

在本发明一实施方式中，所述水凝胶交联前体选自光敏性水凝胶前体、离子型水凝胶前体、热固化水凝胶前体中的一种或几种。优选地，所述水凝胶交联前体为光敏性水凝胶前体和/或离子型水凝胶前体。In one embodiment of the present invention, the hydrogel cross-linking precursor is selected from one or more of photosensitive hydrogel precursors, ionic hydrogel precursors, and thermally curable hydrogel precursors. Preferably, the hydrogel cross-linking precursor is a photosensitive hydrogel precursor and/or an ionic hydrogel precursor.

在本发明一实施方式中，所述热固化水凝胶前体包括但不限于基质胶、聚醚F127二丙烯酸酯中的至少一种。In one embodiment of the present invention, the thermally curable hydrogel precursor includes but is not limited to at least one of Matrigel and polyether F127 diacrylate.

在本发明一实施方式中，所述水凝胶交联前体包括光敏性水凝胶前体、离子型水凝胶前体、基质胶中的一种或几种。In one embodiment of the present invention, the hydrogel cross-linking precursor includes one or more of a photosensitive hydrogel precursor, an ionic hydrogel precursor, and Matrigel.

在本发明一实施方式中，所述水凝胶交联前体包括基质胶，以及任选的光敏性水凝胶前体和/或离子型水凝胶前体。In one embodiment of the present invention, the hydrogel cross-linking precursor includes Matrigel, and optionally a photosensitive hydrogel precursor and/or an ionic hydrogel precursor.

在本发明一实施方式中，所述水凝胶交联前体包括基质胶、光敏性水凝胶前体和离子型水凝胶前体。In one embodiment of the present invention, the hydrogel cross-linking precursor includes Matrigel, a photosensitive hydrogel precursor and an ionic hydrogel precursor.

根据本发明的实施方案，按质量百分比，所述水凝胶交联前体中，所述基质胶的含量≤50%，优选所述基质胶的含量≤40%，进一步优选地，所述基质胶的含量≤30%，例如为35%、25%、20%、18%。According to an embodiment of the present invention, in terms of mass percentage, the content of Matrigel in the hydrogel cross-linking precursor is ≤50%, preferably the content of Matrigel is ≤40%, and further preferably, the matrix The glue content is ≤30%, such as 35%, 25%, 20%, and 18%.

作为一个实例地，按质量百分比，所述水凝胶交联前体中，所述基质胶的含量为20%，光敏性水凝胶前体和/或离子型水凝胶前体的含量为80%。As an example, in terms of mass percentage, in the hydrogel cross-linked precursor, the content of the Matrigel is 20%, and the content of the photosensitive hydrogel precursor and/or the ionic hydrogel precursor is 20%. 80%.

在本发明一实施方式中，所述基质胶包括Matrigel、Cultrex Bme、Geltrex中的一种或几种。In one embodiment of the present invention, the Matrigel includes one or more of Matrigel, Cultrex Bme, and Geltrex.

在本发明一实施方式中，所述水凝胶交联前体包括光敏性水凝胶前体和离子型水凝胶前体，所述光敏性水凝胶前体和离子型水凝胶前体的质量比为1:(0~5)，优选所述光敏性水凝胶前体和离子型水凝胶前体的质量比为1:(0~3)，例如为1:1。In one embodiment of the present invention, the hydrogel cross-linking precursor includes a photosensitive hydrogel precursor and an ionic hydrogel precursor, and the photosensitive hydrogel precursor and the ionic hydrogel precursor The mass ratio of the precursor is 1:(0~5), preferably the mass ratio of the photosensitive hydrogel precursor and the ionic hydrogel precursor is 1:(0~3), for example, 1:1.

在本发明一实施方式中，所述光敏性水凝胶前体包括但不限于甲基丙烯酰化海藻酸钠（AlgMA）、甲基丙烯酰化透明质酸（HAMA）、甲基丙烯酰化壳聚糖、甲基丙烯酰化羧甲基壳聚糖、甲基丙烯酰化聚赖氨酸（PLMA）、甲基丙烯酰化明胶（GM）、甲基丙烯酰化丝素蛋白（SilMA）、甲基丙烯酰化葡聚糖（DeXMA）、甲基丙烯酰化硫酸软骨素（ChSMA）、聚醚F127二丙烯酸酯、聚乙二醇二丙烯酸酯、四臂聚乙二醇丙烯酸酯、以及其他丙烯酰化的材料（如丙烯酰化RGD肽、丙烯酰化聚乙二醇NHS酯）。In one embodiment of the present invention, the photosensitive hydrogel precursor includes but is not limited to methacrylated sodium alginate (AlgMA), methacrylated hyaluronic acid (HAMA), methacrylated Chitosan, methacryloyl carboxymethyl chitosan, methacryloyl polylysine (PLMA), methacryloyl gelatin (GM), methacryloyl silk fibroin (SilMA) , methacrylated dextran (DeXMA), methacrylated chondroitin sulfate (ChSMA), polyether F127 diacrylate, polyethylene glycol diacrylate, four-arm polyethylene glycol acrylate, and Other acrylated materials (such as acrylated RGD peptide, acrylated polyethylene glycol NHS ester).

在本发明一实施方式中，所述离子型水凝胶前体包括但不限于羧甲基纤维素、海藻酸钠、羧甲基壳聚糖。In one embodiment of the present invention, the ionic hydrogel precursor includes but is not limited to carboxymethyl cellulose, sodium alginate, and carboxymethyl chitosan.

在本发明一实施方式中，所述水凝胶交联前体为光敏性水凝胶前体和离子型水凝胶前体的组合物。研究发现，当选择所述组合物作为水凝胶交联前体时，光敏性水凝胶前体和离子型水凝胶前体可以通过调控光固化时间来调控墨水的粘度，进而控制细胞的生长状态；由于离子交联的键合强度要强于光交联的键合强度，加入离子型水凝胶前体可以进一步强化水凝胶的空间骨架强度，提高水凝胶的稳定性，拓宽细胞生长环境的可调控范围。另外，光固化水凝胶前体中的甲基丙烯酰化海藻酸钠、甲基丙烯酰化明胶和甲基丙烯酰化透明质酸，既具有光固化性能，也具有离子固化性能，在使用时，即使不加入离子型水凝胶前体，也可以先进行光固化调控墨水的粘度，再进行离子固化加强凝胶的稳定性，有效调控细胞的生长环境，也就是说，当选择甲基丙烯酰化海藻酸钠、甲基丙烯酰化明胶和甲基丙烯酰化透明质酸作为光敏性水凝胶前体时，即使仅加入光敏性水凝胶前体，也可以达到与同时加入离子型水凝胶前体相同的效果。In one embodiment of the present invention, the hydrogel cross-linking precursor is a composition of a photosensitive hydrogel precursor and an ionic hydrogel precursor. Research has found that when the composition is selected as a hydrogel cross-linking precursor, the photosensitive hydrogel precursor and the ionic hydrogel precursor can regulate the viscosity of the ink by regulating the light curing time, thereby controlling the cell activity. Growth state; since the bonding strength of ionic cross-linking is stronger than that of photo-cross-linking, adding ionic hydrogel precursor can further strengthen the spatial skeleton strength of the hydrogel, improve the stability of the hydrogel, and broaden the cell The controllable range of the growth environment. In addition, methacryloyl alginate, methacryloyl gelatin and methacryloyl hyaluronic acid in the photocurable hydrogel precursor have both photocurable and ion curable properties. Even if the ionic hydrogel precursor is not added, the viscosity of the ink can be controlled by light curing first, and then ionic curing can be performed to enhance the stability of the gel and effectively regulate the growth environment of the cells. In other words, when selecting methyl When acryloyl sodium alginate, methacryloyl gelatin and methacryloyl hyaluronic acid are used as photosensitive hydrogel precursors, even if only the photosensitive hydrogel precursor is added, the same effect as that of adding ions at the same time can be achieved. The same effect as the hydrogel precursor.

在本发明一实施方式中，当所述水凝胶交联前体包括光敏性水凝胶前体时，所述分散介质包括光引发剂。In one embodiment of the present invention, when the hydrogel cross-linking precursor includes a photosensitive hydrogel precursor, the dispersion medium includes a photoinitiator.

在本发明一实施方式中，所述光敏性水凝胶前体与光引发剂的质量比为(2~15):1；优选所述光敏性水凝胶前体与光引发剂的质量比为(3~12):1；例如为4:1、7:1、9:1、10:1、12:1。In one embodiment of the present invention, the mass ratio of the photosensitive hydrogel precursor to the photoinitiator is (2~15):1; preferably the mass ratio of the photosensitive hydrogel precursor to the photoinitiator It is (3~12):1; for example, it is 4:1, 7:1, 9:1, 10:1, 12:1.

在本发明一实施方式中，所述分散介质还至少包括水凝胶粘附增强剂，所述水凝胶粘附增强剂用于增强水凝胶对基底的粘附性。具体的，所述水凝胶粘附增强剂包括但不限于聚多巴胺、聚甲基丙烯酰胺、聚乙二醇、聚乙烯醇等中的至少一种。In one embodiment of the present invention, the dispersion medium further includes at least a hydrogel adhesion enhancer, which is used to enhance the adhesion of the hydrogel to the substrate. Specifically, the hydrogel adhesion enhancer includes but is not limited to at least one of polydopamine, polymethacrylamide, polyethylene glycol, polyvinyl alcohol, etc.

作为一个实例，所述分散介质至少包括羧甲基纤维素和聚多巴胺。As an example, the dispersion medium includes at least carboxymethylcellulose and polydopamine.

作为一个实例，所述分散介质至少包括海藻酸盐、羧甲基纤维素和聚多巴胺。As an example, the dispersion medium includes at least alginate, carboxymethylcellulose and polydopamine.

作为一个实例，所述分散介质至少包括聚多巴胺、光敏性水凝胶前体和光引发剂。As an example, the dispersion medium at least includes polydopamine, a photosensitive hydrogel precursor and a photoinitiator.

作为一个实例，所述分散介质至少包括海藻酸盐、羧甲基纤维素、聚多巴胺和壳聚糖。As an example, the dispersion medium includes at least alginate, carboxymethylcellulose, polydopamine and chitosan.

在本发明一实施方式中，在生物活性墨水中，所述分散介质中至少包括海藻酸盐、羧甲基纤维素、聚多巴胺、光敏性水凝胶前体和引发剂。In one embodiment of the present invention, in the bioactive ink, the dispersion medium at least includes alginate, carboxymethylcellulose, polydopamine, a photosensitive hydrogel precursor and an initiator.

在本发明一具体实施方式中，所述海藻酸盐、羧甲基纤维素、聚多巴胺、光敏性水凝胶前体和引发剂的质量比为(1~5):1:0.5:(1~5):(0.05~0.25)。In a specific embodiment of the present invention, the mass ratio of alginate, carboxymethylcellulose, polydopamine, photosensitive hydrogel precursor and initiator is (1~5):1:0.5:(1 ~5):(0.05~0.25).

示例性地，所述质量比中，海藻酸盐的配比为1~5中的任一点值，具体例如1、2、3、4或5。Illustratively, in the mass ratio, the proportion of alginate is any value from 1 to 5, specifically such as 1, 2, 3, 4 or 5.

示例性地，所述质量比中，光敏性水凝胶前体的配比为1~5中的任一点值，具体例如1、2、3、4或5。Illustratively, in the mass ratio, the proportion of the photosensitive hydrogel precursor is any value from 1 to 5, specifically such as 1, 2, 3, 4 or 5.

示例性地，所述质量比中，引发剂的配比为0.05~0.25中的任一点值，具体例如0.05、0.10、0.15、0.20或0.25。Illustratively, in the mass ratio, the proportion of the initiator is any value from 0.05 to 0.25, specifically such as 0.05, 0.10, 0.15, 0.20 or 0.25.

在本发明一具体实施方式中，所述光敏性水凝胶前体包括甲基丙烯酰化海藻酸钠。In a specific embodiment of the present invention, the photosensitive hydrogel precursor includes methacrylated sodium alginate.

在本发明一具体实施方式中，所述引发剂包括苯基-2,4,6-三甲基苯甲酰基亚磷酸锂(LAP)。In a specific embodiment of the present invention, the initiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphite (LAP).

在本发明一具体实施方式中，所述分散介质中还包括层粘蛋白、RGD多肽、细胞因子、抗生素、小分子化合物、纤维素酶、培养基中的一种或几种。In a specific embodiment of the present invention, the dispersion medium further includes one or more of lamin, RGD polypeptide, cytokines, antibiotics, small molecule compounds, cellulase, and culture medium.

在本发明一具体实施方式中，所述层粘蛋白和/或RGD多肽的浓度为0.1-1mg/ml。In a specific embodiment of the present invention, the concentration of laminin and/or RGD polypeptide is 0.1-1 mg/ml.

在本发明进一步的具体实施方式中，所述细胞因子包括但不限于：R-Spondin细胞生长因子、mNoggin细胞生长因子、EGF细胞生长因子、Wnt3a细胞生长因子中的至少一种。In further specific embodiments of the present invention, the cytokines include but are not limited to: at least one of R-Spondin cell growth factor, mNoggin cell growth factor, EGF cell growth factor, and Wnt3a cell growth factor.

在本发明一具体实施方式中，所述细胞因子的浓度为50-100 ng/mL。In a specific embodiment of the present invention, the concentration of the cytokine is 50-100 ng/mL.

在本发明进一步的具体实施方式中，所述抗生素包括但不限于Primocin™原代细胞抗生素。In further embodiments of the invention, the antibiotics include, but are not limited to, Primocin™ primary cell antibiotics.

在本发明一具体实施方式中，所述抗生素的浓度为50-100μg/ mL。In a specific embodiment of the present invention, the concentration of the antibiotic is 50-100 μg/mL.

在本发明进一步的具体实施方式中，所述小分子化合物包括但不限于SB202190、Gastrin I、A83-01、烟酰胺、Prostaglandin E2、及N-乙酰-L-半胱氨酸中的至少一种。In further specific embodiments of the present invention, the small molecule compounds include but are not limited to at least one of SB202190, Gastrin I, A83-01, nicotinamide, Prostaglandin E2, and N-acetyl-L-cysteine .

在本发明一具体实施方式中，所述小分子化合物的浓度为10 nM-10 mM。In a specific embodiment of the present invention, the concentration of the small molecule compound is 10 nM-10 mM.

在本发明进一步的具体实施方式中，所述纤维素酶包括但不限于内切葡聚糖酶、外切葡聚糖酶、β-葡萄糖苷酶。In further specific embodiments of the present invention, the cellulase includes but is not limited to endoglucanase, exoglucanase, and β-glucosidase.

在本发明一具体实施方式中，所述纤维素酶的浓度为0.01-0.1%。In a specific embodiment of the present invention, the concentration of cellulase is 0.01-0.1%.

在本发明进一步的具体实施方式中，所述培养基包括但不限于DMEM/F12培养基。In further specific embodiments of the present invention, the culture medium includes but is not limited to DMEM/F12 culture medium.

在本发明一实施方式中，所述生物单元包括细胞、组织或类器官。In one embodiment of the invention, the biological unit includes cells, tissues or organoids.

在本发明一具体实施方式中，所述生物单元中细胞的浓度为104-106个/ml。In a specific embodiment of the present invention, the concentration of cells in the biological unit is 10 4 -10 6 /ml.

在本发明一具体实施方式中，所述细胞包含但不限于正常原代细胞、肿瘤原代细胞、细胞系、诱导多能干细胞等中的至少一种。In a specific embodiment of the present invention, the cells include but are not limited to at least one of normal primary cells, tumor primary cells, cell lines, induced pluripotent stem cells, and the like.

在本发明一具体实施方式中，所述组织包含但不限于细胞团、小块生物组织、细胞质基质等中的一种或多种。具体的，所述生物组织包含但不限于上皮组织、结缔组织、肌肉组织、神经组织中的一种或多种。In a specific embodiment of the present invention, the tissue includes but is not limited to one or more of cell clusters, small pieces of biological tissue, cytoplasmic matrix, etc. Specifically, the biological tissue includes but is not limited to one or more of epithelial tissue, connective tissue, muscle tissue, and nervous tissue.

在本发明一具体实施方式中，所述类器官包括但不限于包括小肠类器官、胃类器官、结肠类器官、肺类器官、膀胱类器官、大脑类器官、肝脏类器官、胰腺类器官、肾脏类器官、卵巢类器官、食道类器官、心脏类器官中的一种或多种。In a specific embodiment of the present invention, the organoids include, but are not limited to, small intestinal organoids, gastric organoids, colon organoids, lung organoids, bladder organoids, brain organoids, liver organoids, pancreatic organoids, One or more of kidney organoids, ovarian organoids, esophageal organoids, and cardiac organoids.

在本发明一实施方式中，步骤S2中，所述在基底表面制造微槽(也称微小沟槽)的方法包括光刻法、划片法、激光刻蚀法。In one embodiment of the present invention, in step S2, the method of manufacturing microgrooves (also called microgrooves) on the surface of the substrate includes photolithography, scribing, and laser etching.

在本发明一具体实施方式中，所述微槽的宽度为10-200微米，优选为20-50微米；深度为10-100微米，优选为50微米。In a specific embodiment of the present invention, the width of the microgroove is 10-200 microns, preferably 20-50 microns; the depth is 10-100 microns, preferably 50 microns.

在本发明一具体实施方式中，所述微槽形成的图案包括平行直线形、波浪形、十字交叉形，优选为平行直线形。平行直线形中，相邻微槽之间的距离为40-2000微米，优选为100-200微米。In a specific embodiment of the present invention, the pattern formed by the microgrooves includes parallel straight lines, wavy shapes, and cross shapes, and is preferably parallel straight lines. In the parallel linear shape, the distance between adjacent microgrooves is 40-2000 microns, preferably 100-200 microns.

在本发明一具体实施方式中，所述微槽横截面形状包括但不限于矩形、u形、v形、梯形。In a specific embodiment of the present invention, the cross-sectional shape of the microgroove includes but is not limited to rectangular, U-shaped, V-shaped, and trapezoidal.

在本发明一实施方式中，步骤S3中，所述表面修饰包括将基底表面进行多巴胺修饰。In one embodiment of the present invention, in step S3, the surface modification includes subjecting the substrate surface to dopamine modification.

在本发明一具体实施方式中，所述表面修饰包括以下步骤：In a specific embodiment of the present invention, the surface modification includes the following steps:

(1)将多巴胺盐溶解于缓冲液中，得到多巴胺水溶液；(1) Dissolve the dopamine salt in the buffer to obtain a dopamine aqueous solution;

(2)将预处理的基底浸入多巴胺水溶液中，加热，静置得到修饰的基底。(2) Immerse the pretreated substrate into the dopamine aqueous solution, heat it, and let it stand to obtain the modified substrate.

在本发明进一步的具体实施方式中，所述表面修饰包括以下步骤：In further specific embodiments of the present invention, the surface modification includes the following steps:

a、将多巴胺盐酸盐溶解于Tris-HCl缓冲液(pH=8.8)得到90 mg/mL 的多巴胺水溶液；a. Dissolve dopamine hydrochloride in Tris-HCl buffer (pH=8.8) to obtain a 90 mg/mL dopamine aqueous solution;

b、将基底浸入上述多巴胺水溶液，60℃加热6h后室温静置过夜，移除剩余溶液，PBS清洗，氮气吹干，得到聚多巴胺接枝的高黏附性基底。b. Immerse the substrate into the above-mentioned dopamine aqueous solution, heat at 60°C for 6 hours and then let it stand at room temperature overnight. Remove the remaining solution, wash with PBS, and blow dry with nitrogen to obtain a highly adhesive substrate grafted with polydopamine.

在一些具体实施方式中，所述基底的材料包括聚二甲基硅氧烷膜（PDMS）、聚对苯二甲酸乙二醇酯膜（PET）、ABS塑料膜、聚四氟乙烯膜、透明质酸水凝胶、海藻酸水凝胶、纤维素水凝胶、壳聚糖水凝胶、胶原蛋白水凝胶、明胶水凝胶、丝素水凝胶、琼脂水凝胶、DNA水凝胶、去细胞化组织水凝胶、聚乙烯醇水凝胶、聚丙烯酸水凝胶、聚乙二醇水凝胶、聚多巴胺（PDA）水凝胶或聚丙烯酸酯水凝胶等柔性材料；优选为聚对苯二甲酸乙二醇酯膜或聚多巴胺水凝胶。In some specific embodiments, the material of the substrate includes polydimethylsiloxane film (PDMS), polyethylene terephthalate film (PET), ABS plastic film, polytetrafluoroethylene film, transparent Amino acid hydrogel, alginic acid hydrogel, cellulose hydrogel, chitosan hydrogel, collagen hydrogel, gelatin hydrogel, silk fibroin hydrogel, agar hydrogel, DNA hydrogel , flexible materials such as decellularized tissue hydrogel, polyvinyl alcohol hydrogel, polyacrylic acid hydrogel, polyethylene glycol hydrogel, polydopamine (PDA) hydrogel or polyacrylate hydrogel; preferred It is polyethylene terephthalate film or polydopamine hydrogel.

在一些具体实施方式中，所述的基底的材料包括塑料类、硅类、金属类等一种或多种硬性材料。In some specific embodiments, the material of the substrate includes one or more hard materials such as plastic, silicon, metal, etc.

在一些具体实施方式中，步骤S4中，3D打印的参数设置包括：所述打印图案设置为点阵、线条或2D/3D图案；所述打印针头距离基底的高度为20-100微米；所述单位点喷墨时间为0-10s；所述喷墨气压为0-60 psi，精度为0.1 psi；所述基底接触角为0-100度，优选为50±10度；所述墨滴打印精度为100-1500微米，优选为300-500微米。In some specific embodiments, in step S4, the parameter settings for 3D printing include: the printing pattern is set to a dot matrix, a line or a 2D/3D pattern; the height of the printing needle from the substrate is 20-100 microns; The unit point inkjet time is 0-10s; the inkjet air pressure is 0-60 psi, and the accuracy is 0.1 psi; the substrate contact angle is 0-100 degrees, preferably 50±10 degrees; the ink droplet printing accuracy 100-1500 microns, preferably 300-500 microns.

本发明还提供一种细胞或类器官芯片、特别是一种高粘附性细胞或类器官芯片，其是由上述任一种制备方法制备得到的。The present invention also provides a cell or organoid chip, especially a highly adhesive cell or organoid chip, which is prepared by any of the above preparation methods.

本发明还提供上述细胞或类器官芯片在药物筛选、药物毒性和功效测试、器官模型构建或组织工程中的应用。The present invention also provides the application of the above-mentioned cell or organoid chip in drug screening, drug toxicity and efficacy testing, organ model construction or tissue engineering.

相比于现有技术，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

(1)本发明中通过对所述基底经预处理构造微槽，一方面能够增加基底表面的粗糙度，另一方面能让墨滴与基底形成铆钉作用，从而提高墨滴在基底表面的粘附性。(1) In the present invention, by preprocessing the substrate to construct microgrooves, on the one hand, the roughness of the substrate surface can be increased, and on the other hand, the ink droplets and the substrate can form a riveting effect, thereby improving the adhesion of the ink droplets on the substrate surface. Attachment.

(2)本发明通过对所属基底表面进行化学分子修饰，使墨滴与基底之间形成分子键，通过分子作用力可以显著提高打印物与基底之间的粘附性。(2) In the present invention, the surface of the substrate is modified with chemical molecules to form a molecular bond between the ink droplets and the substrate, and the adhesion between the printed matter and the substrate can be significantly improved through molecular forces.

(3)相比传统手工点样技术，本发明方法所需细胞量少，粘附性高，自动化程度高，极大减少细胞培养成本、培养时间及人工成本，降低人工误差。(3) Compared with traditional manual spotting technology, the method of the present invention requires less cells, high adhesion, and high degree of automation, which greatly reduces cell culture costs, culture time and labor costs, and reduces manual errors.

(4)本发明的芯片可以是一种具有类器官微阵列的药物筛选芯片，所述芯片可以利用少量样本实现药物筛选。(4) The chip of the present invention can be a drug screening chip with an organoid microarray, and the chip can realize drug screening using a small amount of samples.

附图说明Description of the drawings

图1为不同类型阵列型高粘附性细胞或类器官芯片中墨滴的实物图。Figure 1 is a physical picture of ink droplets in different types of array-type highly adhesive cells or organoid chips.

图2为聚多巴胺处理后的基底表面扫描电子显微镜图片。Figure 2 is a scanning electron microscope picture of the substrate surface after polydopamine treatment.

图3为正常细胞生长活力ATP测试结果图。Figure 3 shows the ATP test results of normal cell growth activity.

图4为不同基质胶含量下细胞和类器官在不同生长时间下的SEM图。Figure 4 shows SEM images of cells and organoids at different growth times with different matrigel contents.

图5为不同基质胶含量下细胞和类器官在不同生长时间下的细胞活力测试图。Figure 5 is a graph showing the cell viability test of cells and organoids under different matrigel contents at different growth times.

具体实施方式Detailed ways

下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.

除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

实施例1 细胞培养Example 1 Cell culture

1、细胞分离1. Cell separation

将离体肠癌组织放入1:2碘伏/PBS混合液中浸泡五分钟，用含链霉素和青霉素的PBS清洗五次。清洗后组织放入15 mL 消化液中，剪碎，37 ℃水浴消化40分钟。加入15 mLDMEM/F12培养基后过滤去除无法消化的组织块，滤液1200转离心5分钟，去除上清液。加入10 mL红细胞裂解液裂解10分钟，1200转离心5分钟，去除上清液。用5 mL分离液清洗细胞后，加入适量Matrigel基质胶，吹吸混合后移入24孔板(50 μL每孔)，37℃固化30分钟，加入培养基(500 μL每孔)后放入培养箱中。Soak the isolated intestinal cancer tissue in a 1:2 iodophor/PBS mixture for five minutes, and wash it five times with PBS containing streptomycin and penicillin. After cleaning, the tissue was put into 15 mL of digestive solution, cut into pieces, and digested in a water bath at 37°C for 40 minutes. Add 15 mL of DMEM/F12 culture medium and filter to remove indigestible tissue blocks. The filtrate is centrifuged at 1200 rpm for 5 minutes and the supernatant is removed. Add 10 mL red blood cell lysis solution for lysis for 10 minutes, centrifuge at 1200 rpm for 5 minutes, and remove the supernatant. After washing the cells with 5 mL of separation solution, add an appropriate amount of Matrigel, mix by pipetting and pipetting, then transfer to a 24-well plate (50 μL per well), solidify at 37°C for 30 minutes, add culture medium (500 μL per well), and place in the incubator middle.

上述消化液为添加10 μM Y-27632、100μg/ mL Primocin™原代细胞抗生素、2mg/mL Collagenase Type2酶的分离液。分离液为添加2mM GlutaMAX™、25 mM HEPES、1%链霉素和青霉素的DMEM/F12培养基。The above digestion solution is a separation solution added with 10 μM Y-27632, 100 μg/mL Primocin™ primary cell antibiotic, and 2 mg/mL Collagenase Type2 enzyme. The separation medium is DMEM/F12 medium supplemented with 2mM GlutaMAX™, 25 mM HEPES, 1% streptomycin and penicillin.

2、细胞培养及传代2. Cell culture and passage

细胞在37℃，5% CO2条件下培养。每两天更换培养基，每七天传代。传代步骤如下：每孔加入500μL分离液，移液枪吹吸破碎基质胶后将液体全部转移至离心管。每孔再取1 mL分离液润洗，液体全部转移至上述离心管，2000转离心五分钟。去除上清液，加入TrypLEexpress酶(250 μL每孔)及10 μM Y-27632，37℃水浴消化5分钟。消化后液体用移液枪吹吸数次以进一步破碎大块基质胶后，2000转离心五分钟。去除上清液，加入2 mL分离液重悬后再次2000转离心五分钟。去除上清液，加入适量Matrigel基质胶，吹吸混合后移入24孔板(50μL每孔)，37℃固化30分钟，加入培养基(500μL每孔)后放入培养箱中。提取稳定传代4次后的细胞用于打印及药物筛选。Cells were cultured at 37°C, 5% CO2 . The culture medium was changed every two days and passaged every seven days. The passage steps are as follows: add 500 μL of separation solution to each well, blow with a pipette to break the Matrigel, and transfer all the liquid to a centrifuge tube. Rinse with 1 mL of separation solution from each well, transfer all the liquid to the above-mentioned centrifuge tube, and centrifuge at 2000 rpm for five minutes. Remove the supernatant, add TrypLEexpress enzyme (250 μL per well) and 10 μM Y-27632, and digest in a water bath at 37°C for 5 minutes. After digestion, the liquid was pipetted several times with a pipette to further break up the large pieces of Matrigel, and then centrifuged at 2000 rpm for five minutes. Remove the supernatant, add 2 mL of separation solution, resuspend, and centrifuge again at 2000 rpm for five minutes. Remove the supernatant, add an appropriate amount of Matrigel, mix by pipetting and pipetting, then transfer to a 24-well plate (50 μL per well), solidify at 37°C for 30 minutes, add culture medium (500 μL per well), and place in an incubator. Cells that were stably passaged 4 times were extracted for printing and drug screening.

该步骤中的分离液为添加2mM GlutaMAX™、25 mM HEPES、1%链霉素和青霉素的DMEM/F12培养基。培养基为添加2mM GlutaMAX™、25 mM HEPES、1%链霉素和青霉素、2% B-27™ 添加剂、100 ng/ mL Wnt3a细胞生长因子、1.25 mM N-乙酰基-L-半胱氨酸、500ng/mLR- Spondin、100 ng/ mL mNoggin细胞生长因子、50 ng/mL EGF细胞生长因子、10 nM 胃泌素 I human、0.5 μM A83-01、3 μM SB202190、10 nM 前列腺素 E2、10 mM 烟酰胺、100μg/mL Primocin™原代细胞抗生素的DMEM/F12培养基。The separation medium in this step is DMEM/F12 medium supplemented with 2mM GlutaMAX™, 25 mM HEPES, 1% streptomycin and penicillin. The medium is supplemented with 2mM GlutaMAX™, 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27™ additive, 100 ng/mL Wnt3a cell growth factor, 1.25 mM N-acetyl-L-cysteine , 500ng/mLR-Spondin, 100 ng/mL mNoggin cell growth factor, 50 ng/mL EGF cell growth factor, 10 nM gastrin I human, 0.5 μM A83-01, 3 μM SB202190, 10 nM prostaglandin E2, 10 DMEM/F12 medium with mM nicotinamide and 100 μg/mL Primocin™ primary cell antibiotic.

3、类器官培养3. Organoid culture

调整细胞密度为2~3×106，加入类器官培养液，将培养板放在 37℃ CO2培养箱培养。 每2天更换培养液。Adjust the cell density to 2~3×10 6 , add organoid culture medium, and place the culture plate in a 37°C CO 2 incubator for culture. Change the culture medium every 2 days.

类器官培养基为添加2mM GlutaMAX™、25 mM HEPES、1%链霉素和青霉素、2% B-27™ 添加剂、100 ng/ mL Wnt3a细胞生长因子、1.25 mM N-乙酰基-L-半胱氨酸、500ng/mLR- Spondin、100 ng/ mL mNoggin细胞生长因子、50 ng/mL EGF细胞生长因子、10 nM 胃泌素 I human、0.5 μM A83-01、3 μM SB202190、10 nM 前列腺素 E2、10 mM 烟酰胺、100μg/mL Primocin™原代细胞抗生素的DMEM/F12培养基。Organoid culture medium is supplemented with 2mM GlutaMAX™, 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27™ additive, 100 ng/mL Wnt3a cell growth factor, 1.25 mM N-acetyl-L-cysteine amino acid, 500ng/mLR-Spondin, 100 ng/mL mNoggin cell growth factor, 50 ng/mL EGF cell growth factor, 10 nM gastrin I human, 0.5 μM A83-01, 3 μM SB202190, 10 nM prostaglandin E2 , 10 mM nicotinamide, 100μg/mL Primocin™ primary cell antibiotic in DMEM/F12 medium.

实施例2 配制生物活性墨水中的分散介质Example 2 Preparation of dispersion medium in bioactive ink

配好培养液然后用细菌过滤器除菌。所述培养液为包括RGD多肽 1g、R-Spondin细胞生长因子50mg、SB202190 10μM、纤维素酶0.05%和DMEM/F12培养基1000ml。Prepare the culture medium and then sterilize it with a bacterial filter. The culture medium includes 1g of RGD polypeptide, 50mg of R-Spondin cell growth factor, 10μM of SB202190, 0.05% of cellulase and 1000ml of DMEM/F12 culture medium.

将9000mg的多巴胺盐酸盐溶解于300mL的Tris-HCl（pH=8.8）的缓冲溶液中，用细胞过滤器过滤除菌，然后于60-70℃加热6小时，后静置18小时，过滤后蒸干并研成聚多巴胺粉末。Dissolve 9000 mg of dopamine hydrochloride in 300 mL of Tris-HCl (pH=8.8) buffer solution, filter and sterilize with a cell filter, then heat at 60-70°C for 6 hours, then let stand for 18 hours, filter Evaporate to dryness and grind into polydopamine powder.

配制生物活性墨水的分散介质：将海藻酸钠粉末和羧甲基纤维素粉末平铺，紫外灯下照射3小时灭菌。用上述培养液溶解分散海藻酸钠（NaA）粉末20mg、羧甲基纤维素（CMC）粉末20mg（2%NaA-2%CMC）、聚多巴胺粉末10mg、甲基丙烯酰化海藻酸钠20mg、LAP 1mg，通过搅拌、超声使其均匀溶解，配制得到分散介质。Prepare the dispersion medium of bioactive ink: spread sodium alginate powder and carboxymethylcellulose powder flatly, and sterilize them under UV light for 3 hours. Dissolve and disperse 20 mg of sodium alginate (NaA) powder, 20 mg of carboxymethylcellulose (CMC) powder (2% NaA-2% CMC), 10 mg of polydopamine powder, and 20 mg of methacrylated sodium alginate in the above culture medium. LAP 1mg, dissolve it uniformly by stirring and ultrasonic, and prepare a dispersion medium.

实施例3 制备基底Example 3 Preparation of substrate

1、基底的预处理1. Pretreatment of substrate

将PET基底使用划片机进行沟槽阵列的制备。使用23微米宽的金刚石刀片，深度为50微米，线间距为100、500、1000或2000微米，划片角度为0°和90°。如图1所示多种沟槽的图案及芯片中的墨滴。Use a dicing machine to prepare the trench array on the PET substrate. Use a 23 micron wide diamond blade with a depth of 50 micron, line spacing of 100, 500, 1000 or 2000 micron and scribing angles of 0° and 90°. Figure 1 shows various groove patterns and ink droplets in the chip.

2、基底的修饰2. Modification of the base

将多巴胺盐酸盐溶解于Tris-HCl缓冲液(pH=8.8)得到90 mg/mL的多巴胺水溶液。将预处理的基底浸入上述多巴胺水溶液，60℃加热6h后室温静置过夜，移除剩余溶液，PBS清洗，氮气吹干，得到聚多巴胺接枝的高粘附性基底。如图2所示为聚多巴胺处理后的基底表面扫描电子显微镜图片。Dissolve dopamine hydrochloride in Tris-HCl buffer (pH=8.8) to obtain a 90 mg/mL dopamine aqueous solution. The pretreated substrate was immersed in the above-mentioned dopamine aqueous solution, heated at 60°C for 6 hours, and then left to stand at room temperature overnight. The remaining solution was removed, washed with PBS, and dried with nitrogen to obtain a highly adhesive substrate grafted with polydopamine. Figure 2 shows a scanning electron microscope picture of the substrate surface after polydopamine treatment.

实施例4 制备高粘附性细胞芯片Example 4 Preparation of highly adhesive cell chip

1、制备生物活性墨水1. Preparation of bioactive ink

将实施例1中酶消化后的细胞悬浮液离心，去除上清液，加入实施例2制备的分散介质中，混合均匀后得到生物活性墨水，细胞浓度为105-106/mL。Centrifuge the enzymatically digested cell suspension in Example 1, remove the supernatant, add it to the dispersion medium prepared in Example 2, and mix evenly to obtain a bioactive ink with a cell concentration of 10 5 -10 6 /mL.

2、打印生物活性墨水2. Print bioactive ink

1）将墨管、针头、墨管压力塞等耗材置于高压蒸汽灭菌锅中进行灭菌（130℃，40分钟），打印基底（PET薄膜、孔板）用紫外灯照射30分钟。1) Place ink tubes, needles, ink tube pressure plugs and other consumables in a high-pressure steam sterilizer for sterilization (130°C, 40 minutes), and irradiate the printing substrate (PET film, orifice plate) with UV light for 30 minutes.

2）利用打印设备内置的程序，对打印过程进行编程，包括打印图案坐标设置为点阵、打印针头距离基底的高度50微米、喷墨气压50 psi、单位点喷墨时间0.4s、基底接触角50度、墨滴打印精度350微米。2) Use the built-in program of the printing device to program the printing process, including setting the printing pattern coordinates to a dot matrix, the height of the printing needle from the substrate to 50 microns, the inkjet air pressure to 50 psi, the unit dot inkjet time to 0.4s, and the substrate contact angle. 50 degrees, ink drop printing accuracy of 350 microns.

3)将紫外灯照射后的打印基底平整固定在打印平台上；取300微升生物活性墨水填充于墨管，通过离心除去分散介质中的气泡。将墨管连通点胶机（喷墨气压控制器）与针头，置于移动平台，利用温度控制器控制温度在4℃环境下，运行程序进行打印。打印完成后用1.5%Ca2+（PBS为分散液，pH=7.0）浸泡墨滴10分钟完成固化，制备得到高粘附性细胞芯片。将芯片置于显微镜下观察。结果显示，墨滴成型效果好。3) Fix the printing substrate irradiated by the UV lamp flatly on the printing platform; fill the ink tube with 300 microliters of bioactive ink, and remove air bubbles in the dispersion medium by centrifugation. Connect the ink tube to the dispensing machine (inkjet air pressure controller) and the needle, place it on the mobile platform, use the temperature controller to control the temperature at 4°C, and run the program to print. After printing, soak the ink droplets in 1.5% Ca 2+ (PBS as dispersion, pH=7.0) for 10 minutes to complete solidification, and prepare a highly adhesive cell chip. Place the chip under a microscope and observe it. The results show that the ink droplet forming effect is good.

实施例5 检测芯片中的细胞活性Example 5 Detection of cell activity in the chip

1）对实施例4中打印得到的芯片进行活死细胞免疫染色测试，检验芯片上的墨点中细胞的存活率。1) Perform a living and dead cell immunostaining test on the chip printed in Example 4 to check the survival rate of cells in the ink spots on the chip.

具体方法为：取出样本（即实施例4中打印得到的芯片），用PBS洗涤1-2次，洗去残留的培养基溶液。使用细胞活死染色试剂盒进行染色，先加入足量配制好的碘化丙啶工作液，保证没过细胞，室温孵育10min。去除碘化丙啶工作液，再用足量PBS温和洗涤一遍去除上清。加入足量配制好的钙黄素工作液，保证没过细胞，室温孵育20-45分钟。去除钙黄素工作液，再用足量PBS温和洗涤一遍去除上清。滴加PBS或抗荧光淬灭剂，最后在荧光显微镜下观察细胞活死标记情况。经过统计打印得到的芯片上的墨点中，活细胞的比例为80-95%。The specific method is: take out the sample (ie, the chip printed in Example 4), wash it 1-2 times with PBS, and wash away the remaining culture medium solution. Use a cell live-dead staining kit for staining. First add a sufficient amount of prepared propidium iodide working solution to ensure that the cells are not covered, and incubate at room temperature for 10 minutes. Remove the propidium iodide working solution and wash gently with sufficient PBS to remove the supernatant. Add enough prepared calcein working solution to ensure that the cells are not covered, and incubate at room temperature for 20-45 minutes. Remove the calcein working solution, then wash gently with sufficient PBS to remove the supernatant. Add PBS or anti-fluorescence quenching agent dropwise, and finally observe the cell live and dead markings under a fluorescence microscope. Among the ink dots on the chip obtained through statistical printing, the proportion of viable cells is 80-95%.

2）将实施例4中打印得到的芯片置于类器官培养基中培养，14天后进行活死细胞免疫染色测试，检验墨点中细胞的存活率。培养14天后，墨点中活细胞的比例为85-98%。2) Place the chip printed in Example 4 and culture it in organoid culture medium. After 14 days, perform a live and dead cell immunostaining test to examine the survival rate of the cells in the ink spots. After 14 days of culture, the proportion of viable cells in the ink spots was 85-98%.

3）将实施例4制备的细胞芯片，培养7天，并在第0、1、4、7天分别测试一次芯片中细胞/类器官的ATP量，如图3所示，可知细胞的生长活性较好且逐步增长。3) Culture the cell chip prepared in Example 4 for 7 days, and test the ATP amount of the cells/organoids in the chip on days 0, 1, 4, and 7 respectively. As shown in Figure 3, the growth activity of the cells can be seen. Better and gradually growing.

ATP测试方法：使用CellTiter-Glo® 3D Cell Viability Assay进行细胞活力测试。96孔板中培养的细胞更换培养基，每孔加入100 uL类器官完全培养基。每孔等体积加入100uL CellTiter-Glo® 3D Reagent，充分混合后室温孵育30 min。多功能酶标仪（TECANinfinite M1000 PRO，Swiss）测量化学发光值。ATP test method: Use CellTiter-Glo® 3D Cell Viability Assay for cell viability testing. For the cells cultured in the 96-well plate, the culture medium was replaced, and 100 uL organoid complete culture medium was added to each well. Add 100uL CellTiter-Glo® 3D Reagent in equal volume to each well, mix thoroughly and incubate at room temperature for 30 minutes. A multifunctional microplate reader (TECANinfinite M1000 PRO, Swiss) measured chemiluminescence values.

实施例6 不同比例基质胶对细胞、器官生长的影响Example 6 Effects of different proportions of Matrigel on the growth of cells and organs

按照实施例2的方法配置分散介质，并在分散介质中分别加入质量百分比为5、20、50、的Matrigel基质胶，并设立未添加Matrigel基质胶(0%)、只包括Matrigel基质胶(100%)作为对照。按照实施例4的方法制备高通量细胞芯片，并置于类器官培养基中培养。Configure the dispersion medium according to the method of Example 2, and add Matrigel Matrigel with mass percentages of 5, 20, and 50 respectively in the dispersion medium, and set up a system without adding Matrigel Matrigel (0%) and only including Matrigel Matrigel (100%). %)as comparison. A high-throughput cell chip was prepared according to the method of Example 4 and cultured in organoid culture medium.

观察初始打印后（Day0）和经过7天(Day7)培养后，类器官生长的情况，结果如图4所示。由图4可知，当基质胶的混合比例大于等于50%时，Day0打印细胞密度不均匀，可能发生细胞沉降，经过7天培养后，类器官生长速度快，但是不均匀；当基质胶比例为20%时，Day0打印细胞密度均匀，且培养7天后，类器官生长速度快且均匀度最好；当基质胶比例小于20%时，例如为5%或0%时，Day0打印细胞密度均匀，且培养7天后，类器官生长速度慢且均匀度较差。同时，对细胞培养过程中不同时间细胞活力进行检测。结果如图5所示，D0为初始打印后，D1为第1天，D4为第4天，D7为第7天。由结果可知，基质胶比例为50%时，细胞活力最高，基质胶比例为20%时，细胞活力有所降低。由此可知，当基质胶比例为20%时，细胞生长结构和打印效果均最好。Observe the growth of organoids after initial printing (Day0) and after 7 days of culture (Day7). The results are shown in Figure 4. As can be seen from Figure 4, when the mixing ratio of Matrigel is greater than or equal to 50%, the density of cells printed on Day0 is uneven and cell sedimentation may occur. After 7 days of culture, the organoids grow quickly but unevenly; when the Matrigel ratio is When 20%, the density of Day0 printed cells is uniform, and after 7 days of culture, the organoids grow quickly and have the best uniformity; when the proportion of Matrigel is less than 20%, such as 5% or 0%, the density of Day0 printed cells is uniform, And after 7 days of culture, the organoids grew slowly and had poor uniformity. At the same time, cell viability was detected at different times during the cell culture process. The results are shown in Figure 5. D0 is after the initial printing, D1 is the 1st day, D4 is the 4th day, and D7 is the 7th day. It can be seen from the results that when the Matrigel ratio is 50%, the cell viability is the highest, and when the Matrigel ratio is 20%, the cell viability decreases. It can be seen that when the Matrigel ratio is 20%, the cell growth structure and printing effect are the best.

对比例1Comparative example 1

打印方法同实施例4，不同之处在于：未加入光敏剂The printing method is the same as in Example 4, except that no photosensitizer is added

按照实施例5的方法，检测打印后的墨点中活细胞的比例为50%。According to the method of Example 5, the proportion of viable cells in the printed ink dots was detected to be 50%.

对比例2Comparative example 2

打印方法同实施例4，不同之处在于：使用O2-plasma对基底进行处理，接触角为0-10°。结果：相比使用聚多巴胺处理基底表面，该方法处理的基底表面接触角分布不均匀，且半小时内接触角回复到50-60°；培养细胞过程中，墨点随着培养液的浸泡，逐渐脱落。The printing method is the same as in Example 4, except that O 2 -plasma is used to treat the substrate, and the contact angle is 0-10°. Results: Compared with the use of polydopamine to treat the substrate surface, the contact angle distribution of the substrate surface treated by this method was uneven, and the contact angle returned to 50-60° within half an hour; during the process of culturing cells, the ink dots were soaked in the culture medium. Gradually fall off.

对比例3Comparative example 3

打印方法同实施例4，不同之处在于：基底上没有微槽结构。The printing method is the same as in Example 4, except that there is no micro-groove structure on the substrate.

按照实施例4步骤3的方法，结果显示，墨滴成型效果较差，培养细胞过程中，墨点随着培养液的浸泡，逐渐脱落。According to the method in step 3 of Example 4, the results show that the ink droplet forming effect is poor. During the process of culturing cells, the ink dots gradually fall off as they are soaked in the culture solution.

对比例4Comparative example 4

打印方法同实施例4，不同之处在于：未进行聚多巴胺修饰。The printing method is the same as in Example 4, except that polydopamine modification is not performed.

按照实施例4步骤3的方法，结果显示，墨滴成型效果较差，固化后墨点脱落。According to the method in step 3 of Example 4, the results show that the ink droplet forming effect is poor, and the ink droplets fall off after solidification.

对比例5Comparative example 5

打印方法同实施例4，不同之处在于：使用聚丙烯酰胺对基底进行处理。具体方法为：1600 mg丙烯酰胺、160 mg过硫酸铵、8 mg N, N-亚甲基双丙烯酰溶解于PBS（pH=11），搅拌5min，溶液滴加在基材上（如24孔板每孔300微升）。每孔加入3-5微升离子螯合剂（TMEDA）后于基材表面形成一层高粘度聚丙烯酰胺。后进行打印，打印后进行墨滴固化和培养液浸泡测试，结果为浸泡数小时后，聚丙烯酰胺发生溶胀。The printing method is the same as in Example 4, except that polyacrylamide is used to treat the substrate. The specific method is: 1600 mg acrylamide, 160 mg ammonium persulfate, 8 mg N, N-methylene bisacrylamide are dissolved in PBS (pH=11), stir for 5 minutes, and the solution is dropped on the substrate (such as 24-well 300 µl per well of plate). Add 3-5 microliters of ion chelating agent (TMEDA) to each well to form a layer of high-viscosity polyacrylamide on the surface of the substrate. After printing, the ink droplet curing and culture solution immersion tests were conducted. The results showed that the polyacrylamide swelled after soaking for several hours.

最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified. Modifications or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of the claims of the present invention.

Claims (9)

1. A method for preparing a cell or organoid chip, characterized by: the preparation method comprises the following steps:
s1: preparing a bioactive ink, the bioactive ink comprising a dispersion medium and a biological unit;
s2: manufacturing a micro groove on the surface of a substrate to obtain a pretreated substrate;
s3: carrying out surface modification on the pretreated substrate to obtain a surface modified substrate;
s4: printing the bioactive ink on a substrate with a modified surface by a 3D printing method to obtain the cell or organoid chip;
wherein the dispersion medium at least comprises a hydrogel cross-linking precursor and a hydrogel adhesion enhancer, and the mass ratio of the hydrogel cross-linking precursor to the hydrogel adhesion enhancer is (1-10) 0.5;
the hydrogel crosslinked precursor comprises a photosensitive hydrogel precursor and an ionic hydrogel precursor, wherein the mass ratio of the photosensitive hydrogel precursor to the ionic hydrogel precursor is 1 (0-5); or the hydrogel crosslinking precursor comprises one or more of photosensitive hydrogel precursor, ionic hydrogel precursor and matrigel, wherein the content of the matrigel in the hydrogel crosslinking precursor is less than or equal to 50% by mass;
the dispersion medium also comprises a crosslinking initiator.
2. The method of preparing a cell or organoid chip according to claim 1, wherein: the dispersion medium also comprises one or more of laminin, RGD polypeptide, cytokine, antibiotics, small molecule compound, cellulase and culture medium.
3. The method of preparing a cell or organoid chip according to claim 1, wherein: the biological unit comprises a cell, tissue or organoid.
4. The method of preparing a cell or organoid chip according to claim 1, wherein: in step S2, the method for manufacturing the micro-grooves on the substrate surface includes photolithography, dicing or laser etching.
5. The method of preparing a cell or organoid chip according to claim 1, wherein: in the step S2, the width of the micro groove is 10-200 micrometers, the depth is 10-100 micrometers, and the pattern formed by the micro groove comprises parallel straight lines, waves and crisscross shapes.
6. The method of preparing a cell or organoid chip according to claim 1, wherein: in step S3, the surface modification includes dopamine modification of the surface of the substrate; the concentration of the dopamine is 10-90mg/mL.
7. The method of preparing a cell or organoid chip according to claim 1, wherein: in step S4, the parameter settings of the 3D printing comprise setting the printing pattern as a dot matrix, lines or 2D/3D pattern; the height of the printing needle head from the substrate is 20-100 micrometers; the ink jet air pressure is 0-60 psi, and the precision is 0.1 psi; single-site inkjet time is 0-10s; the contact angle of the substrate is 0-100 degrees; the ink drop printing accuracy is 100-1500 microns.
8. A cell or organoid chip, characterized in that: the chip is prepared by the preparation method of any one of claims 1-7.
9. Use of the cell or organoid chip of claim 8 in drug screening, drug toxicity and efficacy testing, organ model construction or tissue engineering.
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