Preparation method of 3D cell culture body based on sacrificial material, 3D cell culture body and application

Abstract

The invention discloses a preparation method of a 3D cell culture body. The preparation method comprises the following steps: (A) constructing a framework based on a sacrificial material; (B) arranging a first culture cell and a curable biological medium around the skeleton, and then curing the biological medium to form a cured biological medium block; (C) covering the biological medium block with a curable packaging material, then curing the packaging material, and forming a packaging layer on the outer side of the biological medium block; and (D) removing the skeleton so as to form a perfusion structure in the biological medium block, thereby obtaining the 3D cell culture body.

Description

Technical Field

[0001] The present invention relates to biotechnology, and in particular to a preparation method of a 3D cell culture body based on sacrificial materials, the 3D cell culture body and applications. Background Art

[0002] Organoids have been one of the hottest technologies in recent years and have attracted much attention. As a new in vitro 3D cell model, organoids have broad application prospects in many fields such as stem cells and development, regenerative medicine, disease research, drug development and tumor treatment. In addition, compared with organoids, spheroids were once developed as a cell model between 2D cells and organoids. However, compared with traditional 2D culture models, both organoids and spheroid models are preclinical models with good potential. They can partially restore the tissue and molecular characteristics of cells in the body, can simulate organs in the body to a greater extent, and have tissue and organ functions. Organoids can also be combined with in vitro gene editing technology to achieve gene modification at the organ level and realize the study of more gene functions.

[0003] However, at present, neither organoids nor spheroids can completely simulate real tissue organs in terms of morphology, size, and physiological function. This is one of the major bottlenecks in the development of 3D culture technology and has always been a hot topic and difficulty in this research field.

[0004] In vivo, the rapid proliferation of tumors is inseparable from the support of complex vascular networks, which provide oxygen, nutrients and waste exchange. Under the action of chemokines and angiogenic factors produced by autocrine and paracrine mechanisms, vascular endothelial cells migrate to the periphery of tumor cells and proliferate in large numbers, and then develop into blood vessels with the support of tumor stromal cells. In this process, tumor cells and tumor stromal cells interact with each other to form a tumor microenvironment suitable for angiogenesis, and the generated blood vessels further promote the growth and migration of tumors. Therefore, tumor angiogenesis plays an important role in the growth, infiltration and metastasis of tumors, and is also a hot topic in tumor research and the development of new anti-cancer drugs, especially the development of anti-angiogenic drugs. Therefore, whether in terms of overall morphological structure or biological function, the currently cultured single organoid models cannot completely simulate real tumor tissues.

[0005] Although tremendous progress has been made in isolating and culturing cells from native tissues over the past few decades, simple methods to generate tissue structures with physiological cell density maintained by minimal vascular structures remain elusive, making the construction of perfusable vascularized organoid models crucial.

[0006] Overview

[0007] The present invention provides a new method for preparing a 3D culture body with a perfusable vascular network. The perfusable vascular network of the 3D culture body has a capillary network close to the natural state, which can further meet the complex mass transport requirements required by 3D culture.

[0008] As described above, organoid tissues possess complex mass transport requirements, primarily met by blood flow through the multiscale vascular networks of the cardiovascular system. These vessels deliver nutrients and oxygen to all organ systems of the body and remove metabolic byproducts. Furthermore, as engineered tissues evolve toward therapeutically relevant length scales and cell densities, perfusion of these vascular networks to deliver oxygen and nutrients throughout the tissue volume is crucial. Unlike small capillary networks, large perfusable vascular networks can be better controlled using microfluidic technologies. Key to this is the fact that shear stress induced by fluid flow is a key signaling cue for endothelial cells: low levels of shear are required to maintain a healthy monolayer morphology, while high levels of shear are a potent inducer of morphogenesis. Furthermore, seeding these networks with endothelial cells can recapitulate the barrier function and vascular physiology of native vessels. Therefore, strategies to control fluid flow through perfusable vascular networks are essential for generating functional and biologically relevant tissues for regenerative medicine and in vitro physiological modeling.

[0009] While many approaches have been used to fabricate capillary-sized tubular structures and vascular mimics, these models have not been successfully integrated into complete perfusion platforms to date. Despite the versatility of these vessel template approaches, the minimum diameter of engineered vessels integrated into perfusion systems to date has been limited to 150 microns. Strategies based on extrinsic angiogenesis implemented in organ-on-a-chips, and the size of tissues generated, particularly those requiring high blood flow (e.g., brain, liver, heart), have been limited to 400-500 microns in at least one dimension. Due to their small size, the engineered tissues that have been implemented to date do not retain a physiologically relevant signaling environment within the tissue nor do they develop to a level of complexity comparable to in vivo organs.

[0010] To date, bioprinting methods have produced thin tissues that are only viable for short periods of time. To improve their physiological relevance, we have developed a method for bioprinting 3D cell-laden, vascularized tissues. We have successfully produced highly vascularized 3D cultures that have been demonstrated to be over 2 mm thick and can be perfused on a chip for over three months. Their interior is composed entirely of biological media, closely reproducing the physiological state of tissues and organs in the body.

[0011] The present invention provides a new solution to the above technical problems. The details are as follows:

[0012] In one aspect, the present invention provides a method for preparing a 3D cell culture, comprising the following steps:

[0013] (A) Construction of a skeleton based on sacrificial materials;

[0014] (B) disposing first cultured cells and a curable bio-medium around the skeleton, and then curing the bio-medium to form a cured bio-medium block;

[0015] (C) covering the bio-media block with a curable encapsulating material, and then curing the encapsulating material, so that the bio-media block is encapsulated and reinforced;

[0016] (D) Removing the skeleton, thereby forming a perfusion structure inside the biomedia block to obtain the 3D cell culture.

[0017] In some embodiments, the sacrificial material comprises one or more selected from the group consisting of a heat-sensitive material, a photosensitive material, or a combination thereof.

[0018] In some embodiments, the sacrificial material comprises a 30-40% (w / v) Pluronic F-127 solution and / or a 10-15% (w / v) Gelatin solution.

[0019] In some embodiments, at least a portion of the skeleton is constructed by 3D printing.

[0020] In some embodiments, the first cultured cells include target cells; and optionally, one or more cells selected from the group consisting of stromal cells and vascular cells.

[0021] In some embodiments, the target cells include one or more selected from the following group: cardiac tissue cells, brain tissue cells, intestinal tissue cells, gastric tissue cells, pancreatic tissue cells, muscle tissue cells, lung tissue cells, liver tissue cells, kidney tissue cells, spleen tissue cells, or tumor tissue cells.

[0022] In some embodiments, the target cells comprise one or more selected from the group consisting of osteoblasts, chondrocytes, epithelial cells, muscle cells, tumor cells, keratinocytes, glial cells, lung epithelial cells, stem cells, neuronal cells, immune cells, or a combination thereof.

[0023] In some embodiments, the stromal cells are selected from fibroblast stromal cells or mesenchymal stem cells; and the vascular cells are selected from vascular endothelial cells and vascular organoids.

[0024] In some embodiments,

[0025] (i) the proportion of the stromal cells to the first cultured cells is 10%-90%, preferably 20%-70%, more preferably 30%-50%; and / or

[0026] (ii) the ratio of the stromal cells to the target cells in the first cultured cells is: 1:3-10:1; preferably: 1:3-5:1; more preferably 1:2-3:1, more preferably 3:1; and / or

[0027] (iii) the proportion of the vascular cells to the first cultured cells is 10%-90%, preferably 20%-70%, more preferably 30%-50%; and / or

[0028] (iv) The ratio of the vascular cells to the target cells in the first cultured cells is 1:1-10:1; preferably 1:1-5:1; more preferably 1:1-3:1.

[0029] In some embodiments, the stromal cells and the target cells are derived from the same tissue. Optionally, the target tissue cells include tumor tissue cells and the stromal cells include tumor-associated fibroblasts.

[0030] In some embodiments, the biological medium comprises one or more selected from the group consisting of a thermosensitive material, a photosensitive material, or a combination thereof.

[0031] In some embodiments, the biological medium comprises a protein gel, a synthetic gel, a polysaccharide gel and / or a matrix gel.

[0032] In some embodiments, the protein gel comprises one or more selected from the group consisting of collagen I, fibrin, gelatin, elastin, silk, or a combination thereof.

[0033] In some embodiments, the synthetic gel comprises one or more selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol (PVA), (poly(2-hydroxyethyl methacrylate) PHEMA), poly(N-isopropylacrylamide) (PNIPAM), polyethylene oxide (PEO), polyglycolic acid (PGA), or a combination thereof.

[0034] In some embodiments, the polysaccharide gel comprises one or more selected from the group consisting of hyaluronic acid, alginate, chitosan, dextran, and chondroitin sulfate.

[0035] In some embodiments, the matrix gel comprises one or more selected from the group consisting of: Matrigel TM , MaxGelTM, CD-ECM, Tissue-specific dECM Hydrogel, etc.

[0036] In some embodiments, the biological medium further comprises a cell inducer.

[0037] In some embodiments, the cell-inducing agent comprises a cytokine.

[0038] In some embodiments, the first cultured cells comprise lung cancer cells, and the cytokines comprise one or more cytokines selected from the group consisting of EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Y-27632, and Wnt-3a.

[0039] In some embodiments, before step (d), the distance between the center lines of two adjacent segments of the skeleton in the biomedia block does not exceed 2000 microns.

[0040] In some embodiments, the encapsulation material comprises one or more selected from the group consisting of a heat-sensitive material, a photosensitive material, or a combination thereof.

[0041] In some embodiments, the photosensitive material is a photocurable gel.

[0042] In some embodiments, the photocured gel comprises one or more selected from the group consisting of COMA, CSMA, SILMA, GelMA, HAMA, DexMA, or a combination thereof.

[0043] In some embodiments, the encapsulating material has a higher strength than the biological medium after curing; optionally, the encapsulating material has any one or more of the following characteristics: 1. Storage modulus (Storage Modulus) is 10000-200000 [Pa], preferably 20000-190000 [Pa], more preferably 30000-180000 [Pa], more preferably 40000-170000 [Pa], more preferably 50000-160000 [Pa], 60000-150000 [Pa], more preferably 70000-140000 [Pa], more preferably 80000-190000 [Pa], more preferably 100000-180000 [Pa], more preferably 200000-190000 [Pa], more preferably 30000-180000 [Pa], more preferably 40000-170000 [Pa], more preferably 50000-160000 [Pa], 60000-150000 [Pa], more preferably 70000-140000 [Pa], more preferably 80000-180000 [Pa], more preferably 80000-190000 [Pa], more preferably 100000-18 ... 00-120000 [Pa], more preferably 90000-110000 [Pa] and / or 2. loss modulus (Loss Modulus) is 2000-50000 [Pa], preferably 5000-45000 [Pa], more preferably 10000-40000 [Pa], more preferably 20000-30000 [Pa]; optionally, the strength of the encapsulation material after curing is higher than the strength of the biomedium after curing, which means that the storage modulus and / or loss modulus of the biomedium is lower than the storage modulus and / or loss modulus of the encapsulation material; and optionally, the biomedium and / or the encapsulation material is biodegradable.

[0044] In some embodiments, the biological medium is collagen I or Matrigel, and the packaging material is GelMA.

[0045] In some embodiments, the step (D) comprises removing the skeleton at a temperature in the range of 0-37°C. Optionally, the specific temperature is 0-10°C, preferably 3-6°C, and more preferably 4°C.

[0046] In some embodiments, the perfusion structure has a three-dimensional network of channel cavities.

[0047] In some embodiments, the maximum distance between adjacent tubes in the three-dimensional network-shaped tube cavity does not exceed 400 microns.

[0048] In some embodiments, the longest distance from any position of the biomedia block to the perfusion structure does not exceed 200 microns.

[0049] In some embodiments, the diameter of the channel formed by the perfusion structure is 100-2000 microns.

[0050] In some embodiments, it further comprises the following steps:

[0051] (E) seeding second cultured cells in the perfusion structure, so that the second cultured cells adhere to the inner wall of the perfusion structure.

[0052] In some embodiments, the second cultured cells include vascular endothelial cells and tumor-associated fibroblasts.

[0053] In some embodiments, the method further comprises the following steps:

[0054] (G) Continuously perfusing a culture medium into the perfusion structure to allow the target cells to proliferate three-dimensionally in the 3D cell culture, thereby obtaining a proliferated cell mass containing the target cells.

[0055] In some embodiments, the proliferating cell mass contains a newly formed vascular network structure, and the vascular network structure has one or more of the following characteristics:

[0056] i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm;

[0057] ii) have blood vessels expressing CD31 protein;

[0058] iii) having multiple blood vessels interconnected to form a network;

[0059] iv) The vascular network structure is fused with target cells.

[0060] In some embodiments, the proliferating cell aggregate: a) expresses at least one target tissue molecular marker, wherein the target tissue molecular marker is specifically expressed in the target tissue from which the target cells are derived.

[0061] In one aspect, the present invention provides a 3D cell culture, which is prepared by the above preparation method.

[0062] In one aspect, the present invention provides a 3D cell culture in vitro, comprising:

[0063] a) a solidified bio-media block having a three-dimensional network-like perfusion structure inside the bio-media block; and

[0064] b) an encapsulating material covering the outer layer of the solidified biomedia block;

[0065] The inner wall of the perfusion structure has a vascular barrier comprising vascular endothelial cells, and a cell mass comprising target cells is present in a biomedium block adjacent to the perfusion structure. The cell mass contains a newly formed vascular network, and the vascular network has one or more of the following characteristics:

[0066] i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm;

[0067] ii) have blood vessels expressing CD31;

[0068] iii) having multiple blood vessels interconnected to form a network;

[0069] iv) The vascular network structure is fused with target cells.

[0070] In some embodiments, the cell aggregate expresses at least one target tissue molecular marker, and the target tissue molecular marker is specifically expressed in the target tissue from which the target cells are derived.

[0071] In some embodiments, the target cell is a lung cancer cell, and the target tissue is a lung cancer tissue.

[0072] In some embodiments, the target tissue molecular marker includes a lung cancer marker, a lung cancer extracellular matrix marker, or a vascular marker.

[0073] In some embodiments, the cell culture satisfies:

[0074] a) the lung cancer markers include one or more markers selected from the group consisting of CK-7 and TTF-1;

[0075] b) the lung cancer extracellular matrix markers include one or more markers selected from the group consisting of FN, Vimentin, COL1A1, and MMP2;

[0076] d) The vascular markers include: CD31.

[0077] In some embodiments, the cell aggregate simultaneously expresses at least Vimentin, FN, and CK-7.

[0078] In some embodiments, the marker is detected by immunohistochemistry or immunofluorescence.

[0079] In some embodiments, the extracellular matrix markers of lung cancer detected by immunohistochemistry are distributed on the cell membrane.

[0080] In some embodiments, the solidified biomedia block comprises a solidified protein gel, a solidified synthetic gel, a solidified polysaccharide gel and / or a solidified matrix gel.

[0081] In some embodiments, the solidified bio-medium block further comprises: matrix cells and / or cell inducers.

[0082] In some embodiments, the cell-inducing agent comprises a cytokine.

[0083] In some embodiments, the encapsulating material is as defined above.

[0084] In some embodiments, the infused structure is formed by a sacrificial material based approach.

[0085] In some embodiments, the perfusion structure can allow perfusion of culture medium to allow the cell aggregate to proliferate in a three-dimensional manner in the 3D cell culture.

[0086] In some embodiments, the 3D cell culture is capable of allowing at least one cell aggregate to be cultured to at least 1 cubic centimeter.

[0087] In some embodiments, the 3D cell culture is capable of allowing at least one cell aggregate to be cultured continuously for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks.

[0088] In some embodiments, the solidified biomedia block contains at least one cell mass having a size of at least 10 cubic millimeters and having at least one of the following properties a) and b):

[0089] a) expressing the target tissue molecular marker;

[0090] b) at least one region contains a newly formed vascular network having one or more of the following characteristics:

[0091] i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm;

[0092] ii) have blood vessels expressing CD31;

[0093] iii) having multiple blood vessels interconnected to form a network;

[0094] iv) The vascular network structure is fused with target cells.

[0095] In some embodiments, the cell clumps have a size of at least 5 mm3, at least 50 mm3, at least 100 mm3, at least 200 mm3, at least 300 mm3, at least 500 mm3, at least 700 mm3, at least 800 mm3, at least 900 mm3, or at least 1 cm3.

[0096] In some embodiments, the 3D cell culture is prepared by the preparation method of the present invention.

[0097] In one aspect, the present invention provides an in vitro proliferating cell aggregate obtained by culturing the above-mentioned 3D cell culture.

[0098] In one aspect, the present invention provides a 3D cell culture device comprising the above-mentioned 3D cell culture body.

[0099] In some embodiments, the 3D cell culture device further comprises a housing for packaging the 3D cell culture.

[0100] In some embodiments, the 3D cell culture device further comprises a device capable of applying mechanical force to the 3D cell culture.

[0101] In some embodiments, the means for applying a mechanical force is capable of rotating the 3D cell culture.

[0102] In some embodiments, the inlet and / or outlet of the infusion structure is connected to a pump.

[0103] In some embodiments, the pump is controlled by a control system.

[0104] In some embodiments, a bubble removal device is further provided between the inlet and / or outlet of the infusion structure and the pump.

[0105] In some embodiments, the pump and / or the device applying the mechanical force is controlled by a control system.

[0106] In some embodiments, the 3D cell culture device is a microfluidic device.

[0107] In one aspect, the present invention provides a method for testing a drug candidate, comprising:

[0108] a) providing the above-mentioned 3D cell culture body or the above-mentioned 3D cell culture device;

[0109] b) perfusing the candidate drug into the perfusion structure of the 3D cell culture,

[0110] c) measuring the bioactivity of the cell aggregates in the 3D cell culture after the perfusion of the candidate drug to determine the changes compared to the bioactivity before perfusion, and

[0111] d) evaluating the effect of the candidate drug on the biological activity of the cell aggregate based on the change.

[0112] In some embodiments, the cell aggregate comprises tumor cells.

[0113] In some embodiments, the biological activity of the cell aggregate includes proliferation, apoptosis, growth arrest, cell spreading, cell migration, cell-to-cell connection and / or cell morphology change of the cell aggregate.

[0114] In one aspect, the present invention provides a method of culturing a tissue, comprising:

[0115] a) providing the above-mentioned 3D cell culture body or the above-mentioned 3D cell culture device;

[0116] b) perfusing a culture solution into the perfusion structure of the 3D cell culture body to allow the cell aggregate to proliferate in a three-dimensional manner; and

[0117] c) obtaining a proliferated cell mass, wherein the proliferated cell mass contains a newly formed vascularization network, and the vascularization network has one or more of the following characteristics:

[0118] i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm;

[0119] ii) have blood vessels expressing CD31;

[0120] iii) having multiple blood vessels interconnected to form a network;

[0121] v) The vascular network structure is fused with target cells.

[0122] In some embodiments, the method further comprises treating the expanded cell mass so that it is suitable for transplantation into the body.

[0123] As used in this application, the terms "about" and "approximately" are used equivalently. Any numerical value used in this application with or without about / approximately is intended to cover any normal fluctuations understood by a person of ordinary skill in the relevant art.

[0124] Other features, objects and advantages of the present invention will become apparent in the detailed description that follows. However, it should be understood that while the detailed description illustrates embodiments of the present invention, it is provided by way of illustration only and not limitation. Various changes and modifications within the scope of the present invention will be readily apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] The following figures, which together form part of the accompanying drawings, are for illustration purposes only and are not limiting.

[0126] Figure 1 and Figure 2 The lung cancer cell line status on the third day of culture. Figure 1 This is the field of view under a 4x lens. Figure 2This is the field of view under a 10x microscope. Figure 1 a, 2a are A549-GFP (green fluorescence is lung cancer cell line), Figure 1 b, 2b is HUVEC-RFP (red fluorescence indicates vascular endothelial cells, combined with green fluorescence of lung cancer, it can be determined that capillary networks are formed between tumor cell spheres), Figure 1 c and 2c are the cell states under bright field. The black arrows indicate vascular sprouting, further confirming the formation of capillary network structures. Figure 2 d is the organoid vascularized culture structure, where green represents lung cancer tumor organoids and red represents blood vessels formed by HUVEC cells.

[0127] Figure 3 The figure shows the status of lung cancer organoids on the third day of culture (4X), where red arrows represent organoids and green arrows represent CAFs and HUVECs.

[0128] Figure 4 This value represents the marker expression of lung cancer cell lines / lung cancer organoids cultured on day 3 in the 3D culture medium of the present invention. CK7 and TTF-1 are lung cancer marker proteins, MMP2, COL1A1, and FN are extracellular matrix marker proteins, Vimentin is a CAF cell marker, and CD31 is a vascularization-associated marker protein.

[0129] Figure 5 The figure shows HE staining of 3D culture of lung cancer cell lines, in which the nuclei of lung cancer cell lines are colored blue-purple, and the cytoplasm and extracellular matrix are colored red.

[0130] Figure 6 Shows immunohistochemical staining of FN, CD31, CK7, and Vimentin in 3D cultures of lung cancer cell lines.

[0131] Figure 7Figure 3 shows HE staining of 3D lung cancer organoid cultures and immunohistochemical staining for CD31, FN, CK7, and Vimentin. In the HE staining, green arrows indicate cell nuclei; black arrows indicate the cytoplasm and extracellular matrix. In the CD31 staining, red arrows indicate low cytoplasmic and membrane expression of CD31 on days 1 and 3 of culture. In the CK7 staining, blue arrows indicate high cytoplasmic and membrane expression of CK7 on days 1 and 3 of culture. In the FN staining, black arrows indicate low membrane expression of FN on days 1 and 3 of culture. In the Vimentin staining, blue arrows indicate high cytoplasmic and membrane expression of Vimentin on days 1 and 3 of culture. In the Day1 column: the scale bar of the HE image is 100 μm; the scale bar of the CD31, CK7, FN, and Vimentin images is 50 μm; in the Day3 column: the scale bar of the CD31 and CK7 images is 100 μm; the scale bar of the FN and Vimentin images is 50 μm; in the Tissue column: the scale bar of the HE and CK7 images is 200 μm;

[0132] Figure 8 a and Figure 8 b shows an enlarged image of the HE staining of the lung cancer organoid 3D culture.

[0133] in, Figure 8 In a, the yellow arrows represent vascular endothelial cells. The black arrows in the white area represent blood vessels (both large and small, including tiny capillaries). The green arrows indicate tumor tissue or tumor nests. The entire tissue section is from the cultured organoids. This tissue section demonstrates the histological structure of primary lung cancer organoid tumors, which exhibit vascular integration. Scattered cell clumps against the eosin-red background exhibit a histological structure similar to that of native tumors. A fusion of large and small blood vessels / capillaries is present. Figure 8 b is an enlarged HE staining of the tissue section. The black arrows indicate the vascular endothelial cells (analyzed from the morphology and position), and the red arrows indicate the aggregation of tumor nest cells. Figure 9 This is an implementation process of a specific embodiment of the method of the present invention. A shows a top view of the printed skeleton structure, B shows the contents of the gel added during the process, and C shows the entire production process.

[0134] Figure 10 A photo of the microfluidic chip used to print the skeleton is shown. In the photo, 1 represents the magnet for sealing, 2 represents the inlet / outlet for infusion, 3 represents the area for printing the skeleton, which is also the area for culturing cells, 4 represents the upper cover, and 5 represents the base.

[0135] Figure 11The top view of the microfluidic chip used to print the skeleton and its main dimensions are shown. In the figure, 1 represents the magnet for sealing, 2 represents the inlet / outlet for infusion, and 3 represents the area for printing the skeleton, which is also the area for culturing cells.

[0136] Figure 12 The front view of the microfluidic chip used to print the skeleton and its main dimensions are shown. 4 represents the upper cover and 5 represents the base.

[0137] Figure 13 A left-side view of the microfluidic chip used to print the scaffolds is shown, along with its main dimensions.

[0138] Figure 14 An exploded view of the microfluidic chip used to print the scaffold is shown.

[0139] definition

[0140] In order to make the present invention more easily understood, some terms are first defined as follows. The following terms and other terms are further defined throughout the specification.

[0141] About or approximately: As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar to a specified reference value. In certain embodiments, unless otherwise indicated or apparent from the context, the term "about" or "approximately" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a specified reference value in either direction (greater than or less than), except where such values ​​exceed 100% of the possible values.

[0142] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0143] Details

[0144] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0145] Various aspects of the present invention are further described in detail in the following subsections. The use of subsections is not intended to limit the present invention. Each subsection is applicable to any aspect of the present invention. In this application, unless otherwise indicated, the use of "or" means "and / or."

[0146] The present invention provides a method for easily simulating vascularized structures and microenvironments. This method can achieve vascularization of 3D cultures such as organoids. It can also achieve long-term and realistic cultivation of 3D cultures such as organoids by controlling medium flow through microfluidic technology.

[0147] To achieve the above object, the technical solution provided by the present invention is as follows:

[0148] 1. Preparation of 3D cell culture

[0149] In one aspect, the present invention provides a method for preparing a 3D cell culture, comprising the following steps: (A) constructing a sacrificial material-based skeleton; (B) arranging first cultured cells and a curable biomedium around the skeleton, and then curing the biomedium to form a cured biomedium block; (C) covering the biomedium block with a curable encapsulating material, and then curing the encapsulating material to form an encapsulation layer on the outside of the biomedium block; (D) removing the skeleton, thereby forming a perfusion structure within the biomedium block to obtain the 3D cell culture. In some embodiments, the steps are performed in the order of steps (A), (B), (C), and (D).

[0150] sacrificial materials

[0151] In step (A), a skeleton based on a sacrificial material is constructed. As used herein, the term "sacrificial material" refers to a material constructed to form a skeleton for forming a perfusion structure, and a cavity as a perfusion structure can be formed by removing the skeleton. In some embodiments, the sacrificial material has the property of being able to convert between a solid state and a fluid state. In some embodiments, the sacrificial material comprises one or more selected from the following groups: a thermosensitive material, a photosensitive material, or a combination thereof. In some embodiments, the sacrificial material is preferably non-biotoxic. In some embodiments, in some embodiments, the sacrificial material is a thermosensitive material. In some embodiments, the conditions under which the sacrificial material is converted to a fluid state have no significant effect on cultured cells. The sacrificial material comprises Pluronic F127F127 (Pluronic F-127) and / or gelatin. In some embodiments, the sacrificial material comprises 30%-50%, or 30%-40% (w / v) mass concentration of Pluronic F127, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% (w / v) mass concentration of Pluronic F127. In some embodiments, the sacrificial material comprises 5%-15%, or 10%-15% (w / v) mass concentration of gelatin, for example, 10%, 11%, 12%, 13%, 14%, 15% (w / v) mass concentration of gelatin. In some embodiments, the sacrificial material comprises 30%-50%, or 30%-40% (w / v) concentration of Pluronic F127, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% (w / v) mass concentration of Pluronic F127 and / or 5%-15%, or 10%-15% (w / v) mass concentration of gelatin, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% (w / v) mass concentration of gelatin. In some preferred embodiments, the sacrificial material comprises a 40% (w / v) mass concentration of Pluronic F127 and / or a 10% (w / v) mass concentration of gelatin. In this case, the sacrificial material transforms between a liquid and a solid state as the temperature changes, thereby enabling it to be changed from a solid to a liquid state by changing the temperature at an appropriate time, thereby facilitating its removal. More importantly, both Pluronic F127 and gelatin are non-biotoxic, and their morphological transformation rates are relatively fast, and the temperature variation range does not affect cells.

[0152] Used to form a skeleton for the perfusion structure

[0153] In some embodiments, the skeleton for forming the perfusion structure can have any shape. In some embodiments, the shape of the skeleton for forming the perfusion structure is preferably a three-dimensional network structure. In some embodiments, the skeleton for forming the perfusion structure can be formed in any way. In some embodiments, at least a portion of the skeleton for forming the perfusion structure is constructed by 3D printing. In some embodiments, the skeleton for forming the perfusion structure is formed by 3D printing. In some embodiments, the diameter range of the sacrificial material linear structure (i.e., one-dimensional structure) constituting the three-dimensional network structure is between 100-2000 microns, optionally between 200-900 microns, between 300-800 microns, or between 400-600 microns to simulate the diameter of small blood vessels in the human body. In some embodiments, the center point spacing (i.e., line spacing) between adjacent sacrificial material linear structures constituting the three-dimensional network structure is between 1000-2000 microns, optionally between 1100-1400 microns, or between 1200-1300 microns to ensure sufficient material exchange distance. In some embodiments, the overall appearance of the formed three-dimensional network structure can be any desired three-dimensional shape, such as, but not limited to, a cylinder, a cuboid, or a hexagon. In some preferred embodiments, the 3D printer used in the 3D printing process has extrusion printing capabilities. In some embodiments, the 3D printer includes a temperature control module that can adjust the temperature within a range of 0-40°C. In some embodiments, the extrusion needle of the 3D printer has a diameter range of 500-1000 microns. In some embodiments, the 3D printer includes a software module that can control line spacing and layer height. In some embodiments, the print head of the 3D printer is pneumatically controlled. In some specific embodiments, the 3D printing process employs one or more of the following printing conditions: line spacing controlled between 1-1.5 mm; layer height of 0.8 times the diameter of the selected needle; printing speed controlled between 2-3 mm / s; pneumatic pressure controlled between 0.1-0.2 MPa; and printing temperature set between 10-25°C.

[0154] Biological medium and first cultured cells

[0155] In step (B), the first cultured cells and a curable biological medium are configured around the skeleton, and the biological medium is then cured to form a cured biological medium block. As used herein, the term "first cultured cells" refers to the cells configured in step (B). In some embodiments, the first cultured cells include target cells, and the target cells can be any cells that need to be cultured. In some embodiments, the first cultured cells are provided in a non-dissociated form such as a tissue or cell mass containing target cells. For example, the first cultured cells can be provided in the form of organoids. As used herein, the term "organoid" refers to an organoid (Organoids) that is a tissue analog with a certain spatial structure formed by three-dimensional (3D) culture in vitro using adult stem cells, pluripotent stem cells, tumor cells, etc. Organoids can not only be used for disease modeling and drug screening, but also for regenerative medicine. At present, intestinal organoids, salivary gland organoids, etc. have entered clinical trials, and the use prospects of gastric organoids, pancreatic organoids, thyroid organoids, and liver organoids have also been verified.

[0156] The target cells can be obtained by methods well known to those skilled in the art, for example, by collecting them from an organism and / or by differentiating cells with differentiation capacity. In some embodiments, the target cells are collected from a specific tissue of an organism. In some embodiments, the target cells can also be primary cells or cells of an established cell line.

[0157] In some embodiments, the target cells are normal cells and / or disease state cells. In some embodiments, the target cells may include tissue cells of any organ. In some embodiments, the target cells include one or more selected from the group consisting of cardiac tissue cells, brain tissue cells, intestinal tissue cells, gastric tissue cells, pancreatic tissue cells, muscle tissue cells, lung tissue cells, liver tissue cells, kidney tissue cells, spleen tissue cells, tumor tissue cells, skin tissue cells, uterine tissue, bone tissue cells, thyroid tissue, and breast tissue. As used herein, the term "XX (organ name) tissue cells" refers to a cell population having the same or substantially the same biological characteristics as the cell population constituting the specific organ, such a cell population can be obtained, for example, by collecting from an organism and / or by differentiating cells with differentiation ability. In some embodiments, the disease state cells are tumor cells. In some embodiments, the target cells may include cells of any specific type. In some embodiments, the target cells comprise one or more cells selected from the group consisting of osteoblasts, chondrocytes, epithelial cells, muscle cells, tumor cells, keratinocytes, glial cells, lung epithelial cells, stem cells, neurons, immune cells, or combinations thereof. In some embodiments, the target cells comprise tumor cells. In some embodiments, the tumor cells are derived from tumors such as lung cancer, breast cancer, liver cancer, intestinal cancer, gastric cancer, and kidney cancer.

[0158] In some embodiments, the first cultured cells further include other cells, such as stromal cells, to improve the culture environment of the target tissue cells. The addition of these auxiliary cells can further simulate the complex environment surrounding the target tissue. The mixing of multiple cells can more closely mimic the in vivo environment, and the biological factors released between cells can also positively affect their morphology. In some embodiments, the first cultured cells further include one or more cells selected from the group consisting of stromal cells and vascular cells. In some embodiments, the stromal cells are selected from fibroblasts or mesenchymal stem cells. In some embodiments, the stromal cells and / or vascular cells can be of the same or different origin as the target cells. In some embodiments, the stromal cells and the target cells are derived from the same species. In some embodiments, the stromal cells and the target cells are derived from the same individual. In some embodiments, the stromal cells comprise fibroblasts. In some preferred embodiments, the fibroblasts and the target cells are derived from the same tissue sample. In some embodiments, when the target cells comprise tumor cells, the fibroblasts comprise tumor-associated fibroblasts. In some embodiments, the stromal cells account for 10%-90% of the first cultured cells, preferably 20%-70%, and more preferably 30%-50%. In some embodiments, the ratio of the stromal cells to the target cells is 1:3-10:1; preferably 1:3-5:1; more preferably 1:2-3:1, and more preferably 3:1. In some embodiments, the vascular cells and the target cells are derived from the same species. In some embodiments, the vascular cells are selected from vascular endothelial cells or vascular organoids. In some embodiments, the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC cells). In some embodiments, the vascular cells account for 10%-90% of the first cultured cells, preferably 20%-70%, and more preferably 30%-50%. In some embodiments, the ratio of the vascular cells to the target cells is 1:1-10:1; preferably 1:1-5:1; more preferably 1:1-3:1.

[0159] In some embodiments, first cultured cells and a solidifiable biological medium are disposed around the scaffold. In some embodiments, the first cultured cells are dispersed in the biological medium.

[0160] As used herein, the term "biological medium" refers to a medium that can be used for cell culture. The biological medium used in step (B) is a curable biological medium, and its curing mechanism can be any one known in the art, such as light curing, heat curing, vibration-induced crosslinking, etc. In some embodiments, the biotoxicity of the biological medium is lower than that of the encapsulating material. In some preferred embodiments, the biological medium does not have biotoxicity. In some embodiments, the biological medium is biodegradable, so that the extracellular matrix of the cultured cells can replace the original biological medium after long-term cultivation, thereby further simulating the cell microenvironment under the natural environment. In some embodiments, the curing conditions of the biological medium do not have a significant effect on the cultured cells.

[0161] As used herein, the term "biotoxicity" refers to the adverse effects of a material on the growth, differentiation, lifespan, and other vital activities of biological cells. In some embodiments, biotoxicity refers to cytotoxicity, i.e., adverse effects caused by affecting the basic structure and / or physiological processes of cultured cells, leading to disturbances in cell survival, proliferation, and / or function. Biotoxicity or cytotoxicity can be tested by any method known in the art.

[0162] In some embodiments, the biological medium comprises one or more selected from the group consisting of a thermosensitive material, a photosensitive material, or a combination thereof. In some embodiments, the biological medium comprises a protein gel, a synthetic gel, a polysaccharide gel, and / or a matrix gel. In some embodiments, the protein gel comprises one or more selected from the group consisting of collagen I, fibrin, gelatin, elastin, silk, or a combination thereof. In some embodiments, the synthetic gel comprises one or more selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol (PVA), (poly(2-hydroxyethyl methacrylate) PHEMA), poly(N-isopropylacrylamide) (PNIPAM), polyethylene oxide (PEO), polyglycolic acid (PGA), or a combination thereof. In some embodiments, the polysaccharide gel comprises one or more selected from the group consisting of hyaluronic acid, alginate, chitosan, dextran, and chondroitin sulfate. In some embodiments, the matrix gel comprises one or more selected from the group consisting of Matrigel. TM , MaxGelTM, CD-ECM, Tissue-specific dECM Hydrogel, etc.

[0163] In some embodiments, the biological medium further comprises a cell inducer. In some embodiments, the cell inducer comprises a cytokine. As used herein, the term "cell inducer" refers to a molecule with the ability to regulate the growth, differentiation and / or effect of a cell. As used herein, the term "cytokine" refers to a secretory polypeptide or secretory protein with the ability to regulate the growth, differentiation and / or effect of a cell. Generally speaking, it has a smaller molecular weight (e.g., about 5-20 kDa), including but not limited to chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, hormones and growth factors. In some embodiments, the cell inducer is selected from: retinoic acid (RA), TWS119, Cardiogenol AD, IDE1 and IDE2. In some embodiments, the cytokine is selected from EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Wnt-3a. In a specific embodiment, the first cultured cells comprise lung cancer cells, and the cytokines are selected from EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Wnt-3a, and Y-27632.

[0164] In some embodiments, the first cultured cells and the curable biological medium can be configured around the skeleton by any technical method known to technicians. Including but not limited to: injection using a pipette tip, 3D printing, coating, perfusion around the skeleton, etc. In some embodiments, the configuration is performed by applying the biological medium dispersed with the first cultured cells to the skeleton. In some embodiments, the configuration can be performed by applying the biological medium dispersed with the first cultured cells to the skeleton (or the biological medium previously configured and cured). In some embodiments, the application can be performed by coating, etc. In some embodiments, the configuration can be implemented by perfusing the biological medium dispersed with the first cultured cells around the skeleton.

[0165] In some embodiments, the operation of configuring the first cultured cells and the curable biological medium around the skeleton can be performed once or multiple times, and the biological medium, first cultured cells, and / or configuration method used in each configuration can be the same or different. A specific embodiment will be given below for detailed description. It should be noted that the following example is only used to aid understanding and is not intended to limit the specific embodiments or scope of protection of the present invention. For example, the first cultured cells and the curable biological medium can be configured three times in batches. Specifically, in this case, biological medium A can be configured first and cured, followed by biological medium B and cured, and finally biological medium C and cured. Here, A, B, and C are only used to refer to the order in which the biological media are configured, and there is no intention to refer to whether the components of the three are the same or different. Among them, the biological medium A, biological medium B, and biological medium C can be the same or different. Among them, one or both of the biological medium A, biological medium B, and biological medium C may not contain the first cultured cells (at least one biological medium contains the first cultured cells). The biological medium A, biological medium B, and biological medium C may contain the same or different first cultured cells to achieve spatial configuration between different first cultured cells. The configuration methods may be the same or different between multiple configurations. For example, when biological medium A and / or biological medium B are configured by coating, biological medium C may be configured by perfusion.

[0166] Packaging materials

[0167] In step (C), the bio-media block is covered with a curable encapsulating material, and the encapsulating material is then cured to form an encapsulation layer on the outside of the bio-media block. As used herein, the term "encapsulating material" refers to a material that is used to be encapsulated in the outermost layer of a bio-media to support and reinforce the 3D culture and prevent leakage. By encapsulating and reinforcing the encapsulating material, the 3D culture of the present invention can be continuously cultured over a longer period without damage or loss during the process of instilling culture. In some embodiments, the strength of the encapsulating material after curing is higher than the strength of the bio-media after curing, and the strength of the material can be described and detected in a conventional manner in the art. It should be noted that the strength used herein refers to the ability of a material to maintain its form under instilling culture, rather than mechanical strength in the strict sense. In some embodiments, the packaging material has any one or more of the following characteristics: 1. Storage modulus is 10000-200000 [Pa], preferably 20000-190000 [Pa], more preferably 30000-180000 [Pa], more preferably 40000-170000 [Pa], more preferably 50000-160000 [Pa], 60000-1500 00 [Pa], more preferably 70000-140000 [Pa], more preferably 80000-120000 [Pa], more preferably 90000-110000 [Pa]; and / or 2. loss modulus (Loss Modulus) is 2000-50000 [Pa], preferably 5000-45000 [Pa], more preferably 10000-40000 [Pa], more preferably 20000-30000 [Pa]. In some embodiments, the strength of the encapsulating material after curing is higher than the strength of the biomedium after curing means that the storage modulus and / or loss modulus of the biomedium is lower than the storage modulus and / or loss modulus of the encapsulating material. In some embodiments, the storage modulus and loss modulus can be detected by conventional methods in the art. In some embodiments, the storage modulus and loss modulus are obtained by rheometer detection. In some embodiments, the encapsulating material is a curable material, and its curing mechanism can be any one known in the art, such as light curing, heat curing, vibration-induced crosslinking, etc. Preferably, the encapsulating material does not have biotoxicity. In some embodiments, the encapsulating material is biodegradable. In some embodiments, the curing conditions of the encapsulating material have no significant effect on cultured cells. In some embodiments, the encapsulating material comprises one or more selected from the group consisting of a thermosensitive material, a photosensitive material, or a combination thereof. In some embodiments, the photosensitive material is a photocurable gel.In some embodiments, the light-cured gel comprises one or more selected from the group consisting of methacryloylated collagen (COMA), methacryloylated chitosan (CSMA), methacryloylated silk fibroin (SILMA), methacryloylated gelatin (GelMA), hyaluronic acid hydrogel (HAMA), methacryloylated dextran (DexMA), methacryloylated sodium alginate (AlgMA), or combinations thereof. In some embodiments, the biological medium is collagen I or Matrigel. TM , MaxGel™, CD-ECM, Tissue-specific dECM Hydrogel, etc., wherein the encapsulating material is GelMA. In some embodiments, the encapsulating material has a higher strength after curing than the biological medium after curing; and optionally, the biological medium and / or the encapsulating material are biodegradable. In some embodiments, the biological medium is collagen I or Matrigel, and the encapsulating material is GelMA.

[0168] Infused structure after removal of sacrificial material

[0169] In step (D), the skeleton is removed, thereby forming a perfusion structure inside the bio-medium block to obtain the 3D cell culture. As used herein, the term "perfusion structure" refers to a cavity structure formed in the solidified bio-medium block after the skeleton is removed. In some embodiments, the perfusion structure is designed to have one or more inlets and / or outlets for perfusion. It should be noted that the inlets and outlets here can be interchanged according to the specific mode of use. As described in the sacrificial material section, the conditions when the sacrificial material is preferably removed have no significant effect on the cultured cells. For example, when the sacrificial material is a thermosensitive material, the step (D) comprises removing the skeleton within a temperature range of 0-37°C, optionally, the specific temperature is 0-10°C, preferably 3-6°C, and more preferably 4°C. In a preferred embodiment, when the sacrificial material is a thermosensitive material, the process of removing the above-mentioned sacrificial material should be controlled within 30 minutes to avoid causing a significant effect on the cultured cells. It should be noted that the selection of the sacrificial material and the biological medium must satisfy the following requirements: the curing conditions of the biological medium and the removal conditions of the sacrificial material should not be the same or similar, so as to avoid the sacrificial material becoming fluid during the curing process of the biological medium.

[0170] The perfusion structure can have any structure, and the technician can design it arbitrarily according to the perfusion effect that needs to be achieved. In some embodiments, the perfusion structure has a three-dimensional network-shaped pipeline cavity. In some embodiments, the maximum distance between adjacent pipelines in the three-dimensional network-shaped pipeline cavity is no more than 400 microns. In some embodiments, the shortest distance that any position of the biomedia block reaches the perfusion structure is no more than 200 microns. In some embodiments, the diameter of the pipeline formed by the perfusion structure is between 500-1000 microns, optionally, between 600-900 microns, between 700-800 microns. In some embodiments, the diameter of the pipeline formed in the perfusion structure is between 500-1000 microns, optionally, between 600-900 microns, between 700-800 microns.

[0171] Second cultured cells

[0172] In some embodiments, a second cultured cell can be optionally further arranged on the inner wall of the perfusion structure to simulate a desired tissue structure, such as a vascular wall structure, on the inner wall of the perfusion structure. That is, in some embodiments, the method for preparing the 3D cell culture further comprises the following steps: (E) seeding the second cultured cell within the perfusion structure, such that the second cultured cell adheres to the inner wall of the perfusion structure. As used herein, the term "second cultured cell" refers to cells introduced into the perfusion structure through the inlet of the perfusion structure and is used solely to distinguish them from the first cultured cells in terms of their arrangement, and is not intended to indicate whether the first and second cultured cells are identical or different in other aspects, such as composition.

[0173] In some embodiments, by configuring appropriate second culture cells, vascular structures can be further simulated. For example, in some embodiments, the second culture cells include vascular endothelial cells. In some embodiments, the second culture cells further include smooth muscle cells and / or fibroblasts. In some embodiments, the fibroblasts are cancer-associated fibroblasts. In some embodiments, the ratio of the vascular endothelial cells to the cancer-associated fibroblasts is 1:5-5:1; preferably: 3:1-1:3; more preferably 1:2-2:1, more preferably 1:1. In some embodiments, the vascular endothelial cells are provided in the form of vascular organoids or vascular spheres. In some embodiments, the final density of the vascular endothelial cells is 1×10 7 Cells / ml can be seeded at a rate of about 1.0×10 7 pcs, about 2.0×10 7 pcs, about 3.0×10 7 pcs, about 4.0×10 7 pcs, about 5.0×107 pcs, about 6.0×10 7 pcs, about 7.0×10 7 pcs, about 8.0×10 7 pcs, about 9.0×10 7 In some embodiments, the final density of the vascular endothelial cells is 3.0×10 7 Cells are seeded at a density of 100 cells / ml. Too few cells can result in uneven cell attachment, which can lead to ineffective vascular barrier formation and affect subsequent sprouting and the formation of capillary-like structures. Too high a cell density can lead to a large waste of cells and also raise concerns about culture costs. In a preferred embodiment, the cells can be left to stand for 2-6 hours after seeding to allow the second cultured cells to adhere.

[0174] In step (E) and / or after step (E), in order to promote the second cultured cells to adhere to the inner wall of the perfusion structure, technical methods known in the art can be used, for example, adding cell inducers and / or cytokines to the perfusion solution containing the second cultured cells; applying mechanical force, etc.

[0175] In some embodiments, the technical solutions in this field can be further used to promote the growth of vascular endothelial cells to form a vascular barrier for simulating natural vascular structures. As used herein, the term "vascular barrier" refers to a vascular wall-like structure containing vascular endothelial cells. For example, in some embodiments, the preparation method of the 3D cell culture further comprises the following steps: (F) perfusing the perfusion structure with culture medium to allow the vascular endothelial cells to form a vascular barrier on the inner wall of the perfusion structure. In step (F), before step (F) and / or after step (F), in order to form a vascular barrier, technical methods known in the art can be used, for example, adding cell inducers and / or cytokines (such as VEGFA, FGF10, FGF7, Noggin, RSPO1 and Wnt-3a, etc.) to the culture medium; applying mechanical force, etc. Under the porous structure of the bio-medium block, the vascular barrier will invade and sprout into the bio-medium block, and further form a new blood vessel-like structure, which will eventually be intertwined with the cell clumps formed by the proliferation of target cells, thereby simulating the real vascularized microenvironment of the tissue in vivo.

[0176] Culture of target cells

[0177] In some embodiments, the method for preparing the 3D cell culture further comprises the following steps: (G) continuously perfusing the culture medium into the perfusion structure to allow the target cells to proliferate in a three-dimensional manner in the 3D cell culture to obtain a cell mass containing the proliferated target cells. In some embodiments, the culture medium can be perfused at any suitable speed. In some embodiments, the perfusion rate is 1 μl / min-20 μl / min, preferably 3 μl / min-15 μl / min; more preferably 5 μl / min-10 μl / min. As used herein, the term "cell mass" refers to a cell colony obtained by proliferation in a three-dimensional manner. The cell mass can be formed by the proliferation of a cell colony, or it can be formed by the fusion of multiple cell colonies after proliferation.

[0178] The present invention unexpectedly discovered that by implementing steps (A) through (G) above, not only can a 3D cell culture with a perfused structure be obtained, but the newly formed vessel-like structures on the vascular barrier can also fuse and intertwine with the cell aggregate, forming an ideal structure resembling a true vascularized microenvironment. This structure resembles the distribution morphology of natural capillaries, further enhancing the sustainable cultivation capacity of the 3D cell culture, a feat not previously achieved in the prior art. Specifically, in some embodiments, the proliferating cell aggregate contains a newly formed vascular network structure, which exhibits one or more of the following characteristics: i) large vessels with diameters ranging from 10 to 100 microns and capillaries with diameters not exceeding 10 microns; ii) vessels expressing CD31; iii) multiple vessels interconnected to form a network; and iv) the vascular network structure is fused with target cells. In some embodiments, the vascular network structure further exhibits the following characteristics: v) the vascular network structure allows for perfusion culture; in some embodiments, the vascular network structure is fused with target cells. In some embodiments, the vascular network structure is connected to the perfusion structure. As used herein, the term "nascent" refers to structures formed after perfusion culture, rather than structures originally present in the culture material. As used herein, the term "fusion" refers to the close, adherent connection of sprouted tip cells with target cells within a vascular network structure, or the close connection of vascular-like structures with other cells to form a multilayered, organized structure. As used herein, the term "capillary" refers to a vessel-like structure with a diameter not exceeding 10 microns. As used herein, the term "macrovascular" refers to a vessel-like structure with a diameter of 10 to 100 microns relative to capillaries. In some embodiments, the macrovascular structure includes vessel-like structures with a diameter of 10 to 20 microns, a diameter of 20 to 30 microns, a diameter of 30 to 40 microns, a diameter of 40 to 50 microns, a diameter of 50 to 60 microns, a diameter of 60 to 70 microns, a diameter of 70 to 80 microns, and / or a diameter of 80 to 100 microns.

[0179] Similarity to the target organization

[0180] The inventors of the present invention have also unexpectedly discovered that, compared with the prior art, the cell aggregates cultured in the 3D cell culture of the present invention have biological characteristics that are more similar to the target tissue in its natural state. As used herein, the term "target tissue" refers to the tissue in its natural state that the target cells are expected to simulate. Taking organoids as an example, the target tissue of liver organoids is the liver. Taking the "XX (organ name) tissue cells" defined above as an example, the target tissue of "XX (organ name) tissue cells" is XX (organ name) tissue. Without being limited to any theory, the inventors speculate that this is because the vascular-like structure of the present invention and the ideal structure of the fusion of the vascular-like structure and the cell aggregate can be more similar to the natural vascular structure. Biological characteristics refer to various indicators commonly used in the art to characterize the characteristics of biological tissues, including but not limited to genomic characteristics, epigenomic characteristics, transcriptomic characteristics, proteomic characteristics and metabolomic characteristics. In some embodiments, the proliferating cell aggregates: a) express at least one target tissue molecular marker, and the target tissue molecular marker is specifically expressed in the target tissue from which the target cells are derived; and / or b) have gene sequencing results similar to those of the target tissue. In some embodiments, the target tissue molecular markers include extracellular matrix markers of the target tissue, markers of target cells of the target tissue, epithelial-mesenchymal transition markers and / or vascular markers.

[0181] In some embodiments, the target cells are lung cancer, and the target tissue is lung cancer tissue. In some embodiments, the target tissue molecular markers include lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition markers, and / or vascular markers. In some embodiments, a) the lung cancer markers include one or more markers selected from the group consisting of CK-7 and TTF-1; b) the lung cancer extracellular matrix markers include one or more markers selected from the group consisting of FN, COL1A1, MMP2, and Vimentin; and c) the vascular markers include CD31. In one embodiment, the cell aggregates simultaneously express lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition markers, and vascular markers. In one embodiment, the cell aggregates simultaneously express at least FN and Vimentin. Expression of these markers can be detected by methods known in the art, for example, by immunohistochemistry or Western blotting.

[0182] Advantages of the present invention

[0183] The method provided by the present invention can construct multi-layer space vascularized organoids, which can vascularize the surrounding of cultured cells or tissues, so that nutritional factors can be obtained without obstacles and metabolic waste can be discharged in time, thereby simulating the in vivo microenvironment. Previous vascularized gel structures are difficult to achieve the internal space and culture method of such arbitrary structures. In addition, an important content is that the present invention can use a dynamic culture mode, which is different from the traditional completely closed culture. Completely closed culture is very difficult, and it is difficult to achieve long-term culture in many existing models. The present invention can also input the interaction between immune cells and tissues, and the constructed tissue model can break through the limitations of traditional chips and enlarge the size as much as possible.

[0184] The present invention utilizes sacrificial materials to form conduit structures, which are co-printed with printing inks containing biological media, ultimately integrating parenchyma, stroma, and cultured cells into a single thick tissue block. Endothelial cells are then laid into the perfusable conduit structure to form a barrier. These thick tissue blocks can contain growth factors to induce the sprouting of endothelial cells and, optionally, fibroblasts. Ultimately, the capillary network formed within the tissue block and the cultured cell clusters are combined to form a perfusable vascularized cell cluster within the microenvironment. For example, the present invention prints a sacrificial material-based skeleton and uses it as a cell-compatible sacrificial template to generate a network of tubular cavities within an engineered tissue containing living cells. These cavities can be lined with endothelial cells and perfused with culture fluid under high-pressure pulsatile flow. Because this simple vascular casting method allows for independent control of network geometry, endothelialization, and extravascular tissue, it is compatible with a variety of cell types, synthetic and natural extracellular matrices, and cross-linking schemes.

[0185] 2. 3D Cell Culture

[0186] In one aspect, the present invention provides a 3D cell culture, which is prepared by the 3D cell culture preparation method of the present invention.

[0187] In another aspect, the present invention provides a 3D cell culture for in vitro culture, comprising: a) a solidified biomedia block having a three-dimensional network-like perfusion structure within the biomedia block; and b) an encapsulating material covering the outer layer of the solidified biomedia block; wherein the inner wall of the perfusion structure comprises a vascular barrier comprising endothelial cells, and the biomedia block adjacent to the perfusion structure comprises cell clumps comprising target cells, wherein the cell clumps contain newly formed blood vessel-like structures. In some embodiments, the 3D cell culture can be prepared using or without the methods described above for preparing 3D cell cultures.

[0188] In some embodiments, the 3D cell culture contains a newly formed vascular network structure having one or more of the following characteristics: i) large blood vessels with diameters ranging from 10 to 100 microns and capillaries with diameters not exceeding 10 microns; ii) blood vessels expressing CD31; iii) multiple blood vessels interconnected to form a network; and v) the vascular network structure is fused with target cells. In some embodiments, the vascular network structure is fused with target cells. As used herein, the term "fused" refers to the close and adherent connection between the endothelial cells forming the vessel-like structure in the vascular network structure and the target cells. As used herein, the term "capillary" refers to a vessel-like structure with a diameter not exceeding 10 microns. As used herein, the term "large blood vessel" is relative to capillaries and refers to a vessel-like structure with a diameter of 10 to 100 microns. In some embodiments, the large blood vessels comprise vessel-like structures having a diameter between 10 microns and 20 microns, a diameter between 20 microns and 30 microns, a diameter between 30 microns and 40 microns, a diameter between 40 microns and 50 microns, a diameter between 50 microns and 60 microns, a diameter between 60 microns and 70 microns, a diameter between 70 microns and 80 microns, and / or a diameter between 80 microns and 100 microns.

[0189] In some embodiments, the cell aggregates in the 3D cell culture have similarities to the target tissue. The description of "similarity" can refer to the relevant description in the "Similarity to Target Tissue" section. Specifically, in some embodiments, the cell aggregates express at least one target tissue molecular marker, and the target tissue molecular marker is specifically expressed in the target tissue from which the target cells are derived. In some embodiments, the target tissue molecular marker includes an extracellular matrix marker of the target tissue, a marker of target cells of the target tissue, and / or a vascular marker.

[0190] In some embodiments, the target cells are lung cancer, and the target tissue is lung cancer tissue. In some embodiments, the target tissue molecular markers include lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition markers, and / or vascular markers. In some embodiments, the target tissue molecular markers include lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition markers, and / or vascular markers. In some embodiments, a) the lung cancer markers include one or more markers selected from the group consisting of CK-7 and TTF-1; b) the lung cancer extracellular matrix markers include one or more markers selected from the group consisting of FN, COL1A1, MMP2, and Vimentin; and c) the vascular markers include CD31. In one embodiment, the cell aggregates simultaneously express lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition markers, and vascular markers. In one embodiment, the cell aggregates simultaneously express at least FN and Vimentin. Expression of these markers can be performed using methods known in the art. For example, detection can be performed using immunohistochemistry or Western Blot.

[0191] Regarding the concepts of "biological medium" and "solidified biological medium block", the corresponding description in the section "Method for Preparing 3D Cell Culture Body" can be referenced. Specifically, in some embodiments, the solidified biological medium block comprises a solidified protein gel, a solidified synthetic gel, and / or a solidified matrix gel. In some embodiments, the biological medium further comprises a cell inducer. In some embodiments, the cell inducer comprises a cytokine. In some embodiments, regarding the concept of "encapsulation material", the corresponding description in the section "encapsulation material" can be referenced. Regarding the concept of "perfusion structure", the corresponding description in the section "perfusion structure" can be referenced. Regarding the concept of "sacrificial material", the corresponding description in the section "sacrificial material" can be referenced. Specifically, in some embodiments, the perfusion structure is formed using a method based on a sacrificial material. In some embodiments, the perfusion structure can allow perfusion of culture medium to allow the cell clumps to proliferate in a three-dimensional manner in the 3D cell culture body. In some embodiments, the perfusion structure is designed to have one or more inlets and / or outlets for perfusion. It should be noted that the inlets and outlets here can be interchangeable depending on the specific usage.

[0192] The inventors of the present invention unexpectedly discovered that the 3D cell culture of the present invention can overcome the size and duration limitations of the prior art. Specifically, in some embodiments, the 3D cell culture can allow at least one cell cluster to be cultured to at least 10 cubic millimeters. In some embodiments, the 3D cell culture can allow at least one cell cluster to be cultured continuously for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more. Without being limited by theory, the inventors speculate that this is because the vessel-like structures of the present invention, and the ideal structure of the vessel-like structures fused with the cell clusters, are more similar to natural vascular structures and have material exchange capabilities similar to those of natural vascular structures. Specifically, in some preferred embodiments, the 3D cell culture has the following characteristics: the solidified biomaterial block contains at least one cell cluster of at least 10 cubic millimeters in size and exhibiting at least one of the following properties: a) expressing the target tissue molecular marker; and b) at least one region containing capillary-like structures with a diameter of no more than 10 microns. In some preferred embodiments, the size of the cell clump is at least 20 mm3, at least 50 mm3, at least 100 mm3, at least 200 mm3, at least 300 mm3, at least 500 mm3, at least 700 mm3, at least 800 mm3, at least 900 mm3, or at least 1 cm3.

[0193] 3. In vitro proliferation of cell aggregates

[0194] In one aspect, the present invention provides an in vitro proliferating cell aggregate obtained by culturing in the 3D cell culture medium of the present invention. In one embodiment, at least one region of the cell aggregate contains a newly formed vascular network structure, wherein the vascular network structure has one or more of the following characteristics: i) large blood vessels with diameters ranging from 10 to 100 microns and capillaries with diameters not exceeding 10 microns; ii) blood vessels expressing CD31; iii) multiple blood vessels interconnected to form a network; and v) the vascular network structure is fused with target cells. In some embodiments, the vascular network structure is fused with the target cells.

[0195] 4. Culture Device

[0196] The present invention also provides a culture device for continuously culturing the 3D cell culture. Specifically, in one aspect, the present invention provides a 3D cell culture device comprising the 3D cell culture of the present invention. In some embodiments, the 3D cell culture device further comprises a housing for encapsulating the 3D cell culture. The housing can be made of any material, preferably non-biotoxic. In some embodiments, to provide the mechanical force required for cell culture, the 3D cell culture device further comprises a device capable of applying mechanical force to the 3D cell culture. The device for applying mechanical force can be designed to provide the mechanical force required for cell culture or the various tests described below, including but not limited to rotation, vibration, and shaking. Specifically, in some embodiments, the device for applying mechanical force can rotate the 3D cell culture. In some embodiments, the culture device for the 3D cell culture comprises a pump for perfusing a perfusion structure with liquid. In some embodiments, the inlet and / or outlet of the perfusion structure are connected to a pump. In the context of a culture device, the term "pump" refers to any device for transporting or pressurizing a fluid. It should be noted that, for example, the action of the pump may cause bubbles to be contained in the perfused fluid. Due to factors such as shear force when the bubbles burst, this may adversely affect the cultured cells. Therefore, in some preferred embodiments, a bubble removal device is further provided between the inlet and / or outlet of the perfusion structure and the pump. The bubble removal device is not particularly limited, as long as it can eliminate bubbles in the fluid.

[0197] In one aspect, the culture device of the present invention is capable of controllable cell culture. Specifically, in some embodiments, the pump is controlled by a control system. As used herein, the term "control system" refers to a system capable of managing and controlling the main components of the culture device to achieve controllable cell culture. In some embodiments, the control system comprises a computer. In some embodiments, the pump and / or the device for applying mechanical force is controlled by a control system. In some embodiments, the 3D cell culture device is a microfluidic device. As used herein, the term "microfluidic device" refers to a device in which at least one inlet and outlet are interconnected by a microfluidic channel.

[0198] 5. Application

[0199] The 3D cell culture body and / or culture device of the present invention can be efficiently used for three-dimensional cell culture, for example, in organoid preparation, spheroid preparation, three-dimensional tissue culture, etc., and has great application prospects in organoid model construction, animal model construction, drug screening, regenerative medicine, etc.

[0200] The 3D cell cultures of the present invention can be preferably used for anticancer drug screening and drug toxicology testing. For example, organoids have been successfully constructed from primary tumors of the colon, prostate, breast, and pancreas. These "tumor organoids" have become preclinical models that may predict individual patients' responses to treatment. For example, a living biobank of tumor organoids from patients with metastatic gastrointestinal cancer has reproduced the responses of these patients to anticancer drugs in clinical trials. Tumor organoids can also be used to study the tumor niche. A library of organoids representing different grades of colorectal tumors revealed a reduced dependence on niche factors during the transition from normal tissue to adenoma to cancer; it was found that niche factor dependence was mainly related to the genetic composition of the tumor. Tumor organoids are a means of linking cancer-related genomic data with tumor biology, which can provide a basis for drug screening and personalized treatment. For example, organ toxicity is the main reason for drug development failure and post-approval withdrawal. Current toxicology screening using cell lines and animal models is generally unable to predict adverse reactions in humans (of which kidney and liver toxicity are the most common). 3D organoids may provide a more accurate means of predicting toxicity. Currently, kidney organoids have been shown to reproduce the nephrotoxic effects of cisplatin and gentamicin. Other advantages of organoids include their genetic stability and scalability for high-throughput screening. For example, human renal progenitor cells have almost unlimited self-renewal capacity in 3D culture, which may be a boon for the standardization of nephrotoxicity screening. The U.S. Food and Drug Administration (FDA) has begun using three-dimensional "liver chip" organoid models to test the liver toxicity of food additives, nutritional supplements and cosmetics. (Li, Mo, and Juan C. Izpisua Belmonte. "Organoids—Preclinical Models of Human Diseas." New England Journal of Medicine 380.6(2019):569-579.)

[0201] In one aspect, the present invention provides a method for testing a candidate drug, comprising: a) providing the 3D cell culture or the 3D cell culture device; b) perfusing the candidate drug into the perfusion structure of the 3D cell culture, c) measuring the bioactivity of the cell aggregates in the 3D cell culture after the perfusion of the candidate drug to determine the changes compared to the bioactivity before perfusion, and d) evaluating the effect of the candidate drug on the bioactivity of the cell aggregates based on the changes. In some embodiments, the bioactivity of the cell aggregates includes, but is not limited to, proliferation, apoptosis, growth arrest, cell expansion state, cell migration, cell-to-cell connection and / or cell morphology changes of the cell aggregates. In some embodiments, the method is used to screen anti-tumor drugs. In some embodiments, the cell aggregates contain tumor cells.

[0202] In one aspect, the present invention provides a method for culturing tissue, comprising: a) providing the 3D cell culture body or the 3D cell culture device; b) perfusing a culture medium into the perfusion structure of the 3D cell culture body to allow the cell aggregate to proliferate in a three-dimensional manner; and c) obtaining a proliferated cell aggregate, wherein the proliferated cell aggregate contains a blood vessel-like structure. In some embodiments, at least one region of the proliferated cell aggregate contains a newly formed vascular network structure, wherein the vascular network structure has one or more of the following characteristics: i) large blood vessels with a diameter ranging from 10 microns to 100 microns and capillaries with a diameter not exceeding 10 microns; ii) blood vessels expressing CD31; and v) the vascular network structure is fused with target cells. In some embodiments, the vascular network structure is fused with target cells. The tissue can be further used in regenerative medicine. In some embodiments, it further comprises treating the proliferated cell aggregate to make it suitable for transplantation into the body.

[0203] In one aspect, the present invention provides a method for preparing an animal model, comprising: a) providing the 3D cell culture or the 3D cell culture device, wherein the target cells comprise diseased cells from a patient; b) perfusing a culture medium into the perfusion structure of the 3D cell culture to allow the target cells to proliferate in a three-dimensional manner; c) obtaining a proliferated cell mass containing the target cells, wherein the proliferated cell mass contains a capillary-like structure; and

[0204] d) transplanting the proliferated cell mass into an experimental animal to obtain an experimental animal model for the disease.

[0205] In one aspect, the present invention provides uses of the in vitro proliferated cell aggregates, 3D cell cultures, or 3D cell culture devices in animal model preparation, drug testing, and regenerative medicine. As used herein, the term "regenerative medicine" refers to therapeutic methods using cells or cell tissues. In some embodiments, regenerative medicine includes, but is not limited to, tissue engineering and organ engineering. Example

[0206] Example 1. Construction and application of vascularized organoid 3D cultures

[0207] 1.1. Isolation and Culture of Lung Cancer Organoids and Lung Cancer Associated Fibroblasts (LU-CAFs): Lung cancer surgical samples were removed of some blood vessels, adipose tissue, and other tissues using surgical scissors and forceps, and digested with collagenase B (Roche, 11088807001). After digestion, the samples were filtered through a 100 μm filter (Corning, 431752) and centrifuged at 1000 rpm at 4°C for 5 min. After centrifugation, the cells were resuspended in PBS and centrifuged at 1000 rpm at 4°C for 5 min. Finally, red blood cells were lysed using red blood cell lysis buffer (Thermo, A1049201) and centrifuged at 1000 rpm at 4°C for 5 min. Finally, some cell pellets were cultured with Matrigel (Corning, 356231) and cultured in lung cancer organoid-specific culture medium (Xianjuebio). The remaining cell pellets were cultured in the corresponding lung cancer associated fibroblast (LU-CAF) culture medium (Xianjuebio). The culture conditions were 37 degrees Celsius and 5% CO2.

[0208] 1.2. Methods for constructing vascularized organoid 3D cultures:

[0209] 1. Take a 50ml centrifuge tube, add 30ml sterile water, then add 12g Pluronic F127, place in a 4℃ refrigerator for 24h to shake and dissolve, and let it stand for 3 hours to wait for the bubbles to disappear. Finally, prepare a solution with a mass concentration of 40% (w / v) and store in the refrigerator for later use. Weigh 1 g of GelMA, manufactured by Xianjue Biotechnology (methacrylated gelatin), with a degree of substitution of 20%, and place it in a 15 ml centrifuge tube. Add 10 ml of deionized water and then place it in a 50°C water bath to dissolve for 3-4 hours to prepare a 10% GM20 solution. At the same time, use an electronic balance to weigh 150 mg of LAP, manufactured by Xianjue Biotechnology (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate), place it in another 15 ml centrifuge tube, add 3 ml of deionized water, and use a mediator to shake for 10 seconds for later use. After the GelMA is completely dissolved, add 100 μl of LAP solution, followed by 200 μl of 0.1 M NaOH, adjust the pH to 7, and finally filter the GM20 solution using a 0.22 μm filter and place it in a 37°C incubator for later use.

[0210] 2. Quickly remove the Pluronic F127 solution from the refrigerator and slowly load it into the printer's material barrel. Then, load the printer into the printer, turn on the main unit, and select a low-temperature nozzle to print inside the homemade device. Set the syringe temperature to 25°C, the base plate to 30°C, and a 0.5mm diameter long needle. The air pressure parameter is 0.3MPa, the print speed is set to 1mm / s, and the line spacing is 1.5mm. The printing angle is 60°, the print layer height is 0.4mm, and a total of 9 layers are printed. Print the skeleton structure in the central cavity of the microfluidic chip.

[0211] 3. Collect approximately 10,000 lung cancer organoids cultured above, add approximately 30,000 LU-CAFs and 30,000 HUVECs, mix with Matrigel / type I collagen in a 1:1 ratio, and solidify on an F127 scaffold. Finally, fill the interior of the device with the aforementioned Gelma (XJ MATRIX, XJ-GM-20) solution until the entire device chamber is filled. Then, cure with UV light for 3 minutes at an intensity of 10W / cm 2 The device is then sealed

[0212] 4. Transfer the device to a 4°C refrigerator and cool for 20 minutes. After taking it out, use a syringe pump to connect the infusion inlet / outlet on both sides of the chip. Use a one-side infusion and one-side extraction method to remove Pluronic F127 with culture medium at a rate of 20 ml / h.

[0213] 5. Take a piece of HUVEC cells, centrifuge and resuspend in EGM-2 medium to a density of 10 7Use a syringe pump to push HUVEC cells into the perfusable channel from the infusion inlet on one side of the chip at a rate of 1 ml / min. Continue turning the device at a frequency of 90° rotation every 15 minutes for 4 hours until all HUVEC cells are attached to the inner wall of the tube. This step is carried out in a 37°C incubator throughout.

[0214] 6. Use a syringe pump to connect the infusion inlet / outlet on both sides of the above-mentioned chip, and continuously perfuse culture medium at a rate of 5 μl / min. The following cytokines are added to the culture medium to maintain cell and organoid viability, while providing shear force to enable HUVEC cells to form a vascular barrier and keep recording the cell status.

[0215] Example 2. Construction and application of vascularized spheroid 3D culture

[0216] 2.1. Spheroid Formation of Lung Cancer Cell Lines: A549-GFP (Shanghai Chinese Academy of Sciences) cells were cultured in F12K medium (Gibco, 31765035) supplemented with 10% FBS (Gibco, 10099141c); HUVEC / HUVEC-RFP (Zhongqiao Biotechnology) cells were cultured in ECM-specific medium (Sciencell, 1001). All cultures were maintained at 37°C with 5% CO2. Mycoplasma detection was performed using a Mycoplasma Detection Kit (Vazyme / D101-02). Spheroid formation was performed using a low-adhesion 96-well plate (Corning, 7007). A549-GFP and HUVEC-RFP cells were selected for spheroid formation and cultured overnight at 37°C.

[0217] 2.2. Method for constructing 3D culture of vascularized lung cancer cell lines:

[0218] 1. Take a 50ml centrifuge tube, add 30ml sterile water, then add 12g Pluronic F127, place in a 4℃ refrigerator for 24h to shake and dissolve, and let it stand for 3 hours to wait for the bubbles to disappear. Finally, prepare a solution with a mass concentration of 40% (w / v) and store in the refrigerator for later use. Weigh 1 g of GelMA, manufactured by Xianjue Biotechnology (methacrylated gelatin), with a degree of substitution of 20%, and place it in a 15 ml centrifuge tube. Add 10 ml of deionized water and then place it in a 50°C water bath to dissolve for 3-4 hours to prepare a 10% GM20 solution. At the same time, use an electronic balance to weigh 150 mg of LAP, manufactured by Xianjue Biotechnology (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate), place it in another 15 ml centrifuge tube, add 3 ml of deionized water, and use a mediator to shake for 10 seconds for later use. After the GelMA is completely dissolved, add 100 μl of LAP solution, followed by 200 μl of 0.1 M NaOH, adjust the pH to 7, and finally filter the GM20 solution using a 0.22 μm filter and place it in a 37°C incubator for later use.

[0219] 2. Quickly remove the Pluronic F127 solution from the refrigerator and slowly load it into the printer's material barrel. Then, load the printer into the printer, turn on the main unit, and select a low-temperature nozzle to print inside the homemade device. Set the syringe temperature to 25°C, the base plate to 30°C, and a 0.5mm diameter long needle. The air pressure parameter is 0.3MPa, the print speed is set to 1mm / s, and the line spacing is 1.5mm. The printing angle is 60°, the print layer height is 0.4mm, and a total of 9 layers are printed. Print the skeleton structure in the central cavity of the microfluidic chip.

[0220] 3. Collect the mixed spheroids of lung cancer cell line A549-GFP / vascular endothelial cell HUVEC-RFP cultured overnight and add LU-CAF. The cell number ratio is 10,000 lung cancer cells, about 30,000 LU-CAF and 30,000 HUVEC are added. Matrigel / type I collagen is mixed in a ratio of 1:1 and solidified on F127 scaffold.

[0221] 4. Transfer the device to a 4°C refrigerator and cool for 20 minutes. After taking it out, use a syringe pump to connect the infusion inlet / outlet on both sides of the chip. Use a one-side infusion and one-side extraction method to remove Pluronic F127 with culture medium at a rate of 20 ml / h.

[0222] 5. Take a piece of HUVEC cells, centrifuge and resuspend in EGM-2 medium to a density of 10 7Use a syringe pump to push HUVEC cells into the perfusable channel from the infusion inlet on one side of the chip at a rate of 1 ml / min. Continue turning the device at a frequency of 90° rotation every 15 minutes for 4 hours until all HUVEC cells are attached to the inner wall of the tube. This step is carried out in a 37°C incubator throughout.

[0223] 6. Use a syringe pump to connect the infusion inlet / outlet on both sides of the above-mentioned chip, and continuously perfuse culture medium at a rate of 5 μl / min. The following cytokines are added to the culture medium to maintain cell and organoid viability, while providing shear force to enable HUVEC cells to form a vascular barrier and keep recording the cell status.

[0224] The materials and instruments used in the above examples are mainly from the following manufacturers and models:

[0225] The biological 3D printer was purchased from Hangzhou GeneVision Co., Ltd.

[0226] Pluronic F127 was purchased from Sigma Aldrich

[0227] Type I collagen is Type I collagen (rat tail type 1 collagen) was purchased from Corning Incorporated, catalog number: 354236.

[0228] GelMA methacrylated gelatin is a double-bond modified gelatin that can form a hydrogel by UV or visible light crosslinking under the condition of adding a photoinitiator. GelMA hydrogel has excellent biocompatibility and tunable mechanical properties and is widely used in biomedical fields such as tissue engineering, 3D printing, and drug delivery. Purchased from Suzhou Xianjue New Material Technology Co., Ltd.

[0229] All kinds of culture media are produced by our company.

[0230] Example 3. Cultivation and evaluation of 3D cultures

[0231] 3.1 Observe the status of the above-mentioned vascularized lung cancer cell line 3D culture and organoid 3D culture under a microscope.

[0232] The status of lung cancer cell line 3D cultures and organoid 3D cultures on day 3 were observed using a microscope. Figure 1 This is an observation diagram of a 3D culture of a lung cancer cell line. Figure 1 a, 2a are A549-GFP (green fluorescence is lung cancer cell line), Figure 1b, 2b is HUVEC-RFP (red fluorescence indicates vascular endothelial cells, combined with green fluorescence of lung cancer, it can be determined that a capillary-like structure network is formed between tumor cell spheres), Figure 1 c and 2c are the cell states under bright field. The black arrows indicate vascular sprouting, further confirming the capillary-like structural network. Figure 2 (d) shows the vascularized organoid culture structure, with the green representing lung cancer organoids and the red representing blood vessels formed by HUVEC cells. Clear blood vessel branching can be seen, and the vessels are in contact and fused with the tumor organoids.

[0233] Figure 3 This is the state of the organoid 3D culture on the third day of culture.

[0234] 3.2 Western blotting was used to detect the expression of lung cancer, extracellular matrix, epithelial-mesenchymal transition and vascular marker proteins.

[0235] After culturing the above-mentioned 3D culture to the third day, proteins were collected and subjected to immunoblotting experiments. It was found that compared with the 3D cell model of single cell culture (control group: only target cells were cultured without co-culture of HUVEC, CAF and other cells, and other conditions were the same), the vascularized model was able to retain the expression of lung cancer markers itself, and promote the expression of extracellular matrix markers, epithelial-mesenchymal transition markers and vascular markers.

[0236] Figure 4 a and 4b show the results of detecting lung cancer marker proteins (CK7, TTF-1), extracellular matrix marker proteins (MMP2, COL1A1, FN, Vimentin), and vascularization-related marker proteins (CD31) in 3D cultures of lung cancer cell lines and 3D cultures of lung cancer organoids on the third day, respectively.

[0237] It can be seen that compared with the control group, the preparation method of the present invention promotes the expression of ECM proteins, tumor proteins still do not disappear, and promotes the expression of proteins for microvascular formation. Such characteristics are closer to natural tumor tissue.

[0238] 3.3 Hematoxylin / eosin staining (HE staining) was used to observe the morphological structure of vascularized cell lines / organoids.

[0239] Figure 5 The figure shows HE staining of 3D culture sections of lung cancer cell lines, where the nuclei of lung cancer cell lines are blue-purple, and the cytoplasm and extracellular matrix are red. Figure 2 d The tissue structure of fusion between blood vessels and between blood vessels and lung cancer cell tissue can be clearly seen.

[0240] Figure 7The HE row in the middle represents HE staining of lung cancer organoid 3D culture sections. Figure 8 The figure is an enlarged HE staining picture. Figure 8 The yellow arrows in a represent vascular endothelial cells, the black arrows in the white area represent blood vessels (both large and small, and tiny capillaries also exist), and the green arrows represent tumor tissue or tumor nest tissue. The entire tissue section is a section after culturing organoids. This tissue section image confirms that the primary organoid tumor tissue of lung cancer has a tissue structure with vascular fusion. In the eosin-red background, there are some scattered cell clumps, which show a tissue structure similar to that of natural tumors. It can be clearly seen that there is a fusion of large blood vessels and small blood vessels / capillaries, and in the lower part Figure 7 CD31 immunohistochemical staining showed the fusion of tumor tissue and blood vessels, and positive staining of vascular endothelial cells around the cavity structure. Figure 8 b is an enlarged HE staining of a tissue section. The black arrows indicate vascular endothelial cells (analyzed from the perspective of morphology and position), and the red arrows indicate the accumulation of tumor nest cells.

[0241] 3.4 Immunohistochemistry was used to observe the expression of lung cancer, extracellular matrix, epithelial-mesenchymal transition and vascular marker proteins.

[0242] Figure 6 The immunohistochemical results of CD31, CK7, FN, and Vimentin in 3D cultures of lung cancer cell lines are shown. It can be seen that in 3D cultures of lung cancer cell lines, FN is lowly expressed on the cell membrane, CD31 is highly expressed on the cell membrane, and CK7 and Vimentin are highly expressed in the cytoplasm and cell membrane.

[0243] Figure 7 The CD31, CK7, FN, and Vimentin rows in the figure respectively represent the immunohistochemistry results of 3D cultures of lung cancer organoids. It can be seen that in 3D cultures of lung cancer organoids, CD31 is highly expressed on the cell membrane, FN is lowly expressed on the cell membrane, and CK7 and Vimentin are highly expressed in the cytoplasm and cell membrane. A mixed system of tumor and capillary formation can be seen, and in the lumen (vascular-like structure), there is a layer of CD31-positive vascular endothelial cells around it. The other FN, ck7, and Vimentin represent extracellular matrix markers and tumor markers, respectively. This shows that the tumor culture model of the present invention can promote the expression of ECM proteins while tumor proteins do not disappear, and promote the expression of proteins for microvascular formation. Such a tumor culture model can not only be cultured for a long time, but also has biological characteristics similar to the natural state, and can provide an immune microenvironment that is further similar to the natural state.

[0244] Example 4. Specific examples of chip devices

[0245] Figure 9 The following is a schematic diagram of an embodiment of a microfluidic chip and a structure of a fixture, 1 represents the fixture cover, 2 represents the chip cover, 3 represents the chip body, and 4 represents the fixture base. Figure 9 Examples of the preparation and cultivation of 3D cultures using the microfluidic chip shown:

[0246] 1. Take a 50ml centrifuge tube, add 30ml sterile water, then add 12g Pluronic F127, place in a 4℃ refrigerator for 24h to shake and dissolve, and let it stand for 3 hours to wait for the bubbles to disappear. Finally, prepare a solution with a mass concentration of 40% (w / v) and store in the refrigerator for later use. Weigh 1 g of GelMA (methacrylated gelatin, manufactured by Xianjue Biotechnology) with a degree of substitution of 20% and place it in a 15 ml centrifuge tube. Add 10 ml of deionized water and dissolve it in a 50°C water bath for 3-4 hours to prepare a 10% GM20 solution. Simultaneously, use an electronic balance to weigh 150 mg of LAP (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, manufactured by Xianjue Biotechnology) and place it in another 15 ml centrifuge tube. Add 3 ml of deionized water and shake it for 10 seconds using a mediator for later use. After the GelMA is completely dissolved, add 100 μl of LAP solution, followed by 200 μl of 0.1 M NaOH to adjust the pH to 7. Finally, filter the GM20 solution using a 0.22 μm filter and place it in a 37°C incubator for later use.

[0247] 2. Quickly remove the Pluronic F127 solution from the refrigerator and slowly load it into the printer's material barrel. Then, load the printer into the printer, turn on the main unit, and select a low-temperature nozzle to print inside the homemade device. Set the syringe temperature to 25°C, the base plate to 30°C, and a 0.5mm diameter long needle. The air pressure parameter is 0.3MPa, the print speed is set to 1mm / s, and the line spacing is 1.5mm. The printing angle is 60°, the print layer height is 0.4mm, and a total of 9 layers are printed. Print the skeleton structure in the central cavity of the microfluidic chip.

[0248] 3. Collect the mixed spheroids of lung cancer cell line A549-GFP / vascular endothelial cell HUVEC-RFP cultured overnight, add LU-CAF, and solidify them on the F127 scaffold with type I collagen (Corning, 354236). Finally, fill the interior of the device with the above-mentioned Gelma (XJ MATRIX, XJ-GM-20) solution until the entire device chamber is filled. Then use UV light to solidify for 3 minutes at an intensity of 10W / cm 2 The device is then sealed.

[0249] 5. Transfer the device to a 4°C refrigerator and cool for 20 minutes. After taking it out, use a syringe pump to connect the infusion inlet / outlet on both sides of the chip. Use a one-side infusion and one-side extraction method to remove Pluronic F127 with culture medium at a rate of 20 ml / h.

[0250] 6. Take a piece of HUVEC cells, centrifuge and resuspend in EGM-2 medium to a density of 10 7 Use a syringe pump to push HUVEC cells into the perfusable channel from the infusion inlet on one side of the chip at a rate of 1 ml / min. Continue turning the device at a frequency of 90° rotation every 15 minutes for 4 hours until all HUVEC cells are attached to the inner wall of the tube. This step is carried out in a 37°C incubator throughout.

[0251] 7. Use a syringe pump to connect the infusion inlet / outlet on both sides of the above chip and continuously perfuse culture medium at a rate of 5 μl / min to maintain cell and organoid viability. At the same time, provide shear force to enable HUVEC cells to form a vascular barrier and keep recording cell status.

[0252] Equivalent form and scope

[0253] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.It is not intended that the scope of the invention be limited by the above description, but rather by the claims that follow.

[0254] The use of ordinal terms (such as "first," "second," "third," etc.) that modify claim elements in the claims does not in itself imply any priority, superiority, or order of one claim element over another or a temporal order in which method acts are performed, but serves merely as a label to distinguish one claim element having a certain name from another element having the same name (but using ordinal terms), thereby distinguishing the claim elements.

[0255] Unless expressly stated to the contrary, the articles "a" and "an" as used herein in the specification and claims should be understood to include plural referents. Unless the text indicates otherwise or otherwise clearly states, claims or descriptions including "or" between one or more members of a group should be considered to satisfy that one, more than one, or all of the group members are present, employed, or associated with a given product or method. The present invention includes embodiments in which only one member of the group is present, employed, or associated with a given product or method. The present invention also includes embodiments in which more than one or all of the group members are present, employed, or associated with a given product or method. Moreover, it is understood that, unless otherwise stated or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency would arise, the present invention covers all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive modifiers, etc., of one or more of the illustrated claims, introduced into another claim (or any other claim as if related) that is subordinate to the same base claim. When elements are presented as a list (e.g., in a Markush group or similar format), it is understood that it also discloses various subgroups of elements, and any element can be removed from the group. In general, it will be understood that where the present invention or aspects of the present invention include particular elements, features, etc., certain embodiments of the present invention or aspects of the present invention consist of or consist essentially of these elements, features, etc. For the sake of simplicity, those embodiments will not be specifically described in this document with so much verbiage in each case. It will be understood that any embodiment or aspect of the present invention may be explicitly excluded from the claims, regardless of whether such specific exclusion is stated in the specification. Publications, websites, or other reference materials cited herein that describe the background of the present invention and provide additional detailed details about its implementation are incorporated herein by reference.

Claims

1. A method for preparing a 3D cell culture, comprising the following steps: (A) Construction of a skeleton based on sacrificial materials; (B) disposing first cultured cells and a curable bio-medium around the skeleton, and then curing the bio-medium to form a cured bio-medium block; (C) covering the bio-media block with a curable encapsulating material, and then curing the encapsulating material, so that the bio-media block is encapsulated and reinforced; and (D) Removing the skeleton, thereby forming a perfusion structure inside the biomedia block to obtain the 3D cell culture.

2. The method for preparing a 3D cell culture according to claim 1, wherein: The sacrificial material comprises one or more selected from the group consisting of a thermosensitive material, a photosensitive material, or a combination thereof.

3. The method for preparing a 3D cell culture according to claim 1 or 2, wherein: The sacrificial material comprises 30-40% (w / v) Pluronic F-127 solution and / or 10-15% (w / v) Gelatin solution.

4. The method for preparing a 3D cell culture according to any one of claims 1 to 3, wherein: At least a portion of the skeleton is constructed by 3D printing.

5. The method for preparing a 3D cell culture according to any one of claims 1 to 4, wherein: The first cultured cells include target cells; and optionally, one or more cells selected from the following group: stromal cells and vascular cells.

6. The method for preparing a 3D cell culture according to any one of claims 1 to 5, wherein the target cells comprise one or more selected from the group consisting of cardiac tissue cells, brain tissue cells, intestinal tissue cells, gastric tissue cells, pancreatic tissue cells, muscle tissue cells, lung tissue cells, liver tissue cells, kidney tissue cells, spleen tissue cells, or tumor tissue cells.

7. The method for preparing a 3D cell culture according to any one of claims 1 to 6, wherein: The target cells include one or more cells selected from the group consisting of osteoblasts, chondrocytes, epithelial cells, muscle cells, tumor cells, keratinocytes, glial cells, lung epithelial cells, stem cells, neuronal cells, immune cells, or a combination thereof.

8. The method for preparing a 3D cell culture according to any one of claims 1 to 7, wherein: The matrix cells are selected from fibroblast matrix cells or mesenchymal stem cells; the vascular cells are selected from vascular endothelial cells and vascular organoids.

9. The method for preparing a 3D cell culture according to any one of claims 1 to 8, wherein: (i) the proportion of the stromal cells to the first cultured cells is 10%-90%, preferably 20%-70%, more preferably 30%-50%; and / or (ii) the ratio of the stromal cells to the target cells in the first cultured cells is: 1:3-10:1; preferably: 1:3-5:1; more preferably 1:2-3:1, more preferably 3:1; and / or (iii) the proportion of the vascular cells to the first cultured cells is 10%-90%, preferably 20%-70%, more preferably 30%-50%; and / or (iv) The ratio of the vascular cells to the target cells in the first cultured cells is 1:1-10:1; preferably 1:1-5:1; more preferably 1:1-3:

1.

10. The method for preparing a 3D cell culture according to any one of claims 1 to 9, wherein the stromal cells and the target cells are derived from the same tissue, and optionally, the target tissue cells comprise tumor tissue cells and the stromal cells comprise tumor-associated fibroblasts.

11. The method for preparing a 3D cell culture according to any one of claims 1 to 10, wherein: The biological medium comprises one or more selected from the group consisting of a thermosensitive material, a photosensitive material, or a combination thereof.

12. The method for preparing a 3D cell culture according to any one of claims 1 to 11, wherein: The biological medium comprises protein gel, synthetic gel, polysaccharide gel and / or matrix gel.

13. The method for preparing a 3D cell culture according to claim 12, wherein: The protein gel comprises one or more selected from the following group: collagen I, fibrin, gelatin, elastin, silk or a combination thereof.

14. The method for preparing a 3D cell culture according to claim 12, wherein: The synthetic gel comprises one or more selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol (PVA), (poly(2-hydroxyethyl methacrylate) PHEMA), poly(N-isopropylacrylamide) (PNIPAM), polyethylene oxide (PEO), polyglycolic acid (PGA) or a combination thereof.

15. The method for preparing a 3D cell culture according to claim 12, wherein: The polysaccharide gel comprises one or more selected from the following group: hyaluronic acid, alginate, chitosan, dextran, and chondroitin sulfate.

16. The method for preparing a 3D cell culture according to claim 12, wherein: The matrix gel comprises one or more selected from the group consisting of: Matrigel TM 、MaxGel TM , CD-ECM, Tissue-specific dECM Hydrogel, etc.

17. The method for preparing a 3D cell culture according to any one of claims 1 to 16, wherein: The biological medium further comprises a cell inducer.

18. The method for preparing a 3D cell culture according to any one of claims 17, wherein: The cell inducer comprises a cytokine.

19. The method for preparing a 3D cell culture according to any one of claims 1 to 18, wherein: The first cultured cells comprise lung cancer cells, and the cytokines comprise one or more cytokines selected from the group consisting of EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Y-27632, and Wnt-3a.

20. The method for preparing a 3D cell culture according to any one of claims 1 to 19, wherein: Before step (d), the distance between the center lines of two adjacent sections of the skeleton in the bio-media block does not exceed 2000 microns.

21. The method for preparing a 3D cell culture according to any one of claims 1 to 20, wherein: The packaging material comprises one or more selected from the group consisting of a heat-sensitive material, a photosensitive material, or a combination thereof.

22. The method for preparing a 3D cell culture according to any one of claims 2 to 21, wherein: The photosensitive material is a photocurable gel.

23. The method for preparing a 3D cell culture according to claim 22, wherein: The photocurable gel comprises one or more selected from the group consisting of COMA, CSMA, SILMA, GelMA, HAMA, DexMA, or a combination thereof.

24. The method for preparing a 3D cell culture according to any one of claims 1 to 23, wherein the encapsulation material has a higher strength than the biological medium after solidification; optionally, the encapsulation material has any one or more of the following characteristics:

1. a storage modulus of 10,000-200,000 [Pa], preferably 20,000-190,000 [Pa], more preferably 30,000-180,000 [Pa], more preferably 40,000-170,000 [Pa], more preferably 50,000-160,000 [Pa], 60,000-150,000 [Pa], more preferably 70,000-140,000 [Pa], more preferably 80,000-120,000 [Pa], more preferably 90,000-110,000 [Pa] and / or 2. a loss modulus of 10,000-20,000 [Pa], more preferably 10,000-25,000 [Pa], more preferably 15,000-30,000 [Pa], more preferably 16,000-30,000 [Pa], more preferably 17,000-30,000 [Pa], more preferably 18,000-20,000 [Pa], more preferably 20,000-30,000 [Pa], more preferably 21,000-23,000 [Pa], more preferably 23,000-24,000 [Pa], Modulus) is 2000-50000 [Pa], preferably 5000-45000 [Pa], more preferably 10000-40000 [Pa], more preferably 20000-30000 [Pa]; optionally, the strength of the encapsulating material after curing is higher than the strength of the biological medium after curing, which means that the storage modulus and / or loss modulus of the biological medium is lower than the storage modulus and / or loss modulus of the encapsulating material; and optionally, the biological medium and / or the encapsulating material is biodegradable.

25. The method for preparing a 3D cell culture according to any one of claims 1 to 24, wherein: The biological medium is collagen I or matrigel, and the packaging material is GelMA.

26. The method for preparing a 3D cell culture according to any one of claims 1 to 25, wherein: The step (D) comprises removing the skeleton at a temperature in the range of 0-37°C. Optionally, the specific temperature is 0-10°C, preferably 3-6°C, and more preferably 4°C.

27. The method for preparing a 3D cell culture according to any one of claims 1 to 26, wherein: The perfusion structure has a three-dimensional network-shaped pipeline cavity.

28. The method for preparing a 3D cell culture according to any one of claims 1 to 27, wherein: The maximum distance between adjacent pipes in the three-dimensional network-shaped pipe cavity does not exceed 400 microns.

29. The method for preparing a 3D cell culture according to any one of claims 1 to 28, wherein: The longest distance from any position of the biomedia block to the perfusion structure does not exceed 200 microns.

30. The method for preparing a 3D cell culture according to any one of claims 1 to 29, wherein: The diameter of the pipe formed by the perfusion structure is 100-2000 microns.

31. The method of any one of claims 1 to 30, further comprising the steps of: (E) seeding second cultured cells in the perfusion structure, so that the second cultured cells adhere to the inner wall of the perfusion structure.

32. The method for preparing a 3D cell culture according to claim 31, wherein: The second cultured cells include vascular endothelial cells and tumor-associated fibroblasts.

33. The method of any one of claims 1 to 32, further comprising the steps of: (G) Continuously perfusing a culture medium into the perfusion structure to allow the target cells to proliferate three-dimensionally in the 3D cell culture, thereby obtaining a proliferated cell mass containing the target cells.

34. The method of claim 33, wherein the proliferating cell mass contains a newly formed vascular network structure, and the vascular network structure has one or more of the following characteristics: i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm; ii) have blood vessels expressing CD31 protein; iii) having multiple blood vessels interconnected to form a network; iv) The vascular network structure is fused with target cells.

35. The method of any one of claims 33-34, wherein the proliferating cell aggregate: a) expresses at least one target tissue molecular marker, wherein the target tissue molecular marker is specifically expressed in the target tissue from which the target cells are derived.

36. A 3D cell culture, prepared by the preparation method according to any one of claims 1 to 35.

37. A 3D cell culture in vitro, comprising: a) a solidified bio-media block having a three-dimensional network-like perfusion structure inside the bio-media block; and b) an encapsulating material covering the outer layer of the solidified biomedia block; in, The inner wall of the perfusion structure has a vascular barrier containing vascular endothelial cells, and a cell mass containing target cells is provided in a biomedium block adjacent to the perfusion structure. The cell mass contains a newly formed vascular network, and the vascular network has one or more of the following characteristics: i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm; ii) have blood vessels expressing CD31; iii) having multiple blood vessels interconnected to form a network; iv) The vascular network structure is fused with target cells.

38. The 3D cell culture of claim 36 or 37, wherein the cell aggregates express at least one target tissue molecular marker, and the target tissue molecular marker is specifically expressed in the target tissue from which the target cells are derived.

39. The 3D cell culture of any one of claims 36-38, wherein the target cells are lung cancer cells and the target tissue is lung cancer tissue.

40. The 3D cell culture of claim 39, wherein the target tissue molecular marker comprises a lung cancer marker, a lung cancer extracellular matrix marker, or a vascular marker.

41. The 3D cell culture of claim 40, wherein: a) the lung cancer markers include one or more markers selected from the group consisting of CK-7 and TTF-1; b) the lung cancer extracellular matrix markers include one or more markers selected from the following group: FN, Vimentin, COL1A1, MMP2; and / or c) The vascular markers include: CD31.

42. The 3D cell culture according to claim 41, wherein The cell aggregates simultaneously express at least Vimentin, FN and CK-7.

43. The 3D cell culture according to any one of claims 38 to 42, wherein The markers were detected by immunohistochemistry or immunofluorescence.

44. The 3D cell culture according to claim 43, wherein Lung cancer extracellular matrix markers detected by immunohistochemistry were distributed on the cell membrane.

45. The 3D cell culture according to any one of claims 36 to 44, wherein The solidified biological medium block comprises solidified protein gel, solidified synthetic gel, solidified polysaccharide gel and / or solidified matrix gel.

46. ​​The 3D cell culture according to any one of claims 36 to 45, wherein The solidified biological medium block further comprises matrix cells and / or cell inducers.

47. The 3D cell culture according to claim 46, wherein The cell inducer comprises a cytokine.

48. The 3D cell culture according to any one of claims 36 to 47, wherein The encapsulation material is as defined in any one of claims 20-24.

49. The 3D cell culture according to any one of claims 36 to 48, wherein The potting structure is formed by a sacrificial material based approach.

50. The 3D cell culture according to any one of claims 36 to 49, wherein The perfusion structure may allow perfusion of culture medium to allow the cell aggregate to proliferate in a three-dimensional manner in the 3D cell culture.

51. The 3D cell culture according to any one of claims 36 to 50, wherein The 3D cell culture can allow at least one cell cluster to be cultured to at least 1 cubic centimeter.

52. The 3D cell culture according to any one of claims 36 to 51, wherein The 3D cell culture can allow at least one cell aggregate to be cultured continuously for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks.

53. The 3D cell culture according to any one of claims 36 to 52, wherein: The solidified biomedia block contains at least one cell mass having a size of at least 10 cubic millimeters and having at least one of the following properties a) and b): a) expressing the target tissue molecular marker; b) at least one region contains a newly formed vascular network having one or more of the following characteristics: i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm; ii) have blood vessels expressing CD31; iii) having a plurality of blood vessels interconnected to form a network; and / or iv) The vascular network structure is fused with target cells.

54. The 3D cell culture of any one of claims 36-53, wherein the cell clumps have a size of at least 5 mm3, at least 50 mm3, at least 100 mm3, at least 200 mm3, at least 300 mm3, at least 500 mm3, at least 700 mm3, at least 800 mm3, at least 900 mm3, or at least 1 cm3.

55. The 3D cell culture according to any one of claims 36 to 54, which is prepared by the preparation method according to any one of claims 1 to 33.

56. An in vitro proliferated cell aggregate obtained by culturing the 3D cell culture medium according to any one of claims 36 to 55.

57. A 3D cell culture device, comprising the 3D cell culture body according to any one of claims 36 to 55.

58. The 3D cell culture device according to claim 57, wherein The 3D cell culture device further comprises a housing for packaging the 3D cell culture.

59. The 3D cell culture device according to claim 57 or 58, wherein The 3D cell culture device further comprises a device capable of applying mechanical force to the 3D cell culture.

60. The 3D cell culture device according to claim 59, wherein The means for applying a mechanical force is capable of rotating the 3D cell culture.

61. The 3D cell culture device according to any one of claims 57 to 60, wherein: The inlet and / or the outlet of the infusion structure is connected to a pump.

62. The 3D cell culture device of claim 61, wherein: The pump is controlled by a control system.

63. The 3D cell culture device according to any one of claims 61 or 62, wherein: A device for removing air bubbles is further provided between the inlet and / or outlet of the infusion structure and the pump.

64. The 3D cell culture device of any one of claims 61 to 63, wherein: The pump and / or the means for applying a mechanical force are controlled by a control system.

65. The 3D cell culture device of any one of claims 57 to 64, wherein: The 3D cell culture device is a microfluidic device.

66. A method for testing a drug candidate, comprising: a) providing the 3D cell culture body according to any one of claims 36 to 55 or the 3D cell culture device according to any one of claims 57 to 65; b) perfusing the candidate drug into the perfusion structure of the 3D cell culture, c) measuring the bioactivity of the cell aggregates in the 3D cell culture after the perfusion of the candidate drug to determine the changes compared to the bioactivity before perfusion, and d) evaluating the effect of the candidate drug on the biological activity of the cell aggregate based on the change.

67. The method of claim 66, wherein the cell aggregate comprises tumor cells.

68. The method of claim 66 or 67, wherein the biological activity of the cell aggregate comprises proliferation, apoptosis, growth arrest, cell spreading state, cell migration, cell-to-cell connection and / or cell morphology change of the cell aggregate.

69. A method of culturing tissue, comprising: a) providing the 3D cell culture body according to any one of claims 36 to 55 or the 3D cell culture device according to any one of claims 57 to 65; b) perfusing a culture solution into the perfusion structure of the 3D cell culture body to allow the cell aggregate to proliferate in a three-dimensional manner; and c) obtaining a proliferated cell mass, wherein the proliferated cell mass contains a newly formed vascularization network, and the vascularization network has one or more of the following characteristics: i) having large blood vessels with diameters ranging from 10 μm to 100 μm and capillaries with diameters not exceeding 10 μm; ii) have blood vessels expressing CD31; iii) having multiple blood vessels interconnected to form a network; v) The vascular network structure is fused with target cells.

70. The method of claim 69, further comprising treating the expanded cell mass to render it suitable for transplantation into the body.

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