Method for preparing 3D cell culture construct on basis of sacrificial material, and 3D cell culture construct and use thereof

Abstract

Disclosed in the present invention is a method for preparing a 3D cell culture construct on the basis of a sacrificial material, which method comprises the following steps: (A) constructing a scaffold on the basis of a sacrificial material; (B) configuring a first culture cell and a curable biological medium around the scaffold, and then curing the biological medium to form a cured biological medium block; (C) covering the biological medium block with a curable encapsulating material, and then curing the encapsulating material to form an encapsulating layer on the outer side of the biological medium block; and (D) removing the scaffold, thereby forming a perfusion structure inside the biological medium block to obtain the 3D cell culture body.

Description

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

[0001] The present application relates to biotechnology, in particular to a preparation method of 3D cell culture body based on sacrificial material, 3D cell culture body and application. BACKGROUND

[0002] Organoids have been a hot topic in recent years and have attracted much attention. As a new 3D cell model in vitro, organoids have wide 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 have also been developed as a cell model between 2D cells and organoids. However, compared with traditional 2D culture mode, both organoids and spheroids are good potential preclinical models, which can partially restore the tissue and molecular characteristics of cells in vivo, can simulate the in vivo organ to a greater extent, and have the function of tissue organ. Organoids can also be combined with in vitro gene editing technology to realize gene modification at the organ level and realize more gene function research.

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

[0004] In vivo, the rapid proliferation of tumors cannot be separated from the support of complex vascular networks. These vascular networks provide oxygen, nutrients and waste exchange. Under the action of chemotactic factors and pro-angiogenic factors produced by vascular endothelial cells under autocrine and paracrine mechanisms, the vascular endothelial cells migrate around the tumor cells and proliferate in large quantities, and then develop into blood vessels under 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 tumor growth, infiltration and metastasis, and is a hot topic in tumor research and the development of new anti-cancer drugs, especially the development of anti-angiogenic drugs. Therefore, neither the overall morphological structure nor the biological function of the currently cultured single organoid model can completely simulate the real tumor tissue,

[0005] Although great progress has been made in the past few decades in isolating and culturing cells from natural tissues, a simple method to produce tissue structures with physiological cell density maintained by the most basic vascular structure is still elusive, so it is crucial to construct a perfusable vascularized organoid model.

[0006] SUMMARY

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

[0008] As described above, the organoid tissue has complex mass transport requirements, which are mainly met by blood through the multi-scale vascular network of the cardiovascular system. These blood vessels transport nutrients and oxygen to all organ systems of the body and remove metabolic byproducts, and it is essential to deliver oxygen and nutrients to the entire tissue body through perfusion of the vascular network as the engineered tissue develops to the length scale and cell density related to therapy. Different from the small size capillary network, the large size perfusable vascular network can be better controlled by microfluidic technology, the key of which is that the shear stress brought by fluid flow is a key signal for vascular endothelial cells, and low level of shear is required to maintain the healthy monolayer morphology, while high level of shear is an effective inducer of its morphology. In addition, the implantation of endothelial cells in these networks can reproduce the barrier function and vascular physiology of natural blood vessels. Therefore, the strategy of controlling fluid flow through perfusable vascular networks is indispensable for producing functional and biologically relevant tissues for regenerative medicine and in vitro physiological modeling.

[0009] Although many methods have been used to manufacture tubular structures and vascular analogs of capillary size, these models have not been successfully integrated into a complete perfusion platform to date. Although these container template methods have versatility, the minimum diameter of the engineered container integrated into the perfusion system to date has been limited to 150 microns. While implementing the strategy of exogenous angiogenesis in organ-on-a-chip and generating tissues, the size of the generated tissues, especially those that require a large amount of blood flow (such as the brain, liver, and heart), is limited to 400-500 microns in at least one dimension. Due to their small volume, the engineered tissues implemented to date cannot retain a physiologically relevant signaling environment within the tissue, nor can they develop to the complexity comparable to in vivo organs.

[0010] To date, bioprinting methods have produced thin tissues that can only survive for a short period of time. In order to improve their physiological relevance, we invented a method for bioprinting 3D cell-laden, vascularized tissues, and successfully obtained a highly vascularized 3D culture body, which has been proven to have a thickness of more than 2 mm and can be perfused and cultured on a chip for more than 3 months, and its entire interior is composed of biological medium, greatly reducing the physiological state of in vivo tissue organs.

[0011] The present application provides a new solution to the above technical problems. Specifically as follows:

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

[0013] (A) constructing a scaffold based on a sacrificial material;

[0014] (B) disposing first culture cells and a solidifiable biomedia around the scaffold, and then solidifying the biomedia to form a solidified biomedia block;

[0015] (C) covering the biomedia block with a solidifiable encapsulation material, and then solidifying the encapsulation material so that the biomedia block is encapsulated and reinforced;

[0016] (D) removing the scaffold, 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 light-sensitive 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 scaffold is constructed by 3D printing.

[0020] In some embodiments, the first culture cells comprise target cells; and optionally one or more cells selected from the group consisting of: stromal cells and vascular-like cells.

[0021] In some embodiments, 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.

[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-like cells are selected from vascular endothelial cells, vascular-like organs.

[0024] In some embodiments,

[0025] the stromal cells comprise 10-90%, preferably 20-70%, more preferably 30-50% of the first cultured cells; and / or

[0026] the stromal cells comprise 10-90%, preferably 20-70%, more preferably 30-50% of the first cultured cells; and / or

[0027] the vascular cells comprise 10-90%, preferably 20-70%, more preferably 30-50% of the first cultured cells; and / or

[0028] the vascular cells comprise 10-90%, preferably 20-70%, more preferably 30-50% of the first cultured cells; and / or

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

[0030] In some embodiments, the biological medium comprises one or more selected from the group consisting of: a heat-sensitive material, a light-sensitive material, or a combination thereof.

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

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

[0033] In some embodiments, the synthetic-based gel comprises one or more selected from the group consisting of: polyethylene glycol, polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), poly(N-isopropyl acrylamide), poly(ethylene oxide), poly(glycolic acid), or a combination thereof.

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

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

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

[0037] In some embodiments, the cell inducer comprises a cytokine.

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

[0039] In some embodiments, prior to step (D), the spacing between the centerlines of adjacent segments of the scaffold in the block of the biological medium is no more than 2000 microns.

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

[0041] In some embodiments, the light-sensitive material is a photocured 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 encapsulation material has a higher strength after curing than the biological medium after curing; optionally, the encapsulation material has any one or more of the following characteristics:

[0044] a storage modulus of 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 80000-120000 Pa, more preferably 90000-110000 Pa, and / or

[0045] a loss modulus of 2000-50000 Pa, preferably 5000-45000 Pa, more preferably 10000-40000 Pa, more preferably 20000-30000 Pa; optionally, the encapsulation material has a higher strength after curing than the biological medium after curing 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 encapsulation material; and optionally, the biological medium and / or the encapsulation material is biodegradable.

[0046] In some embodiments, the biological medium is collagen I or Matrigel, and the encapsulating material is GelMA.

[0047] In some embodiments, step (D) comprises removing the scaffold at a temperature ranging from 0-37°C, optionally, the specific temperature is 0-10°C, preferably 3-6°C, more preferably 4°C.

[0048] In some embodiments, the perfusion structure has a three-dimensional network of channel cavities.

[0049] In some embodiments, the maximum distance between adjacent channels in the three-dimensional network of channel cavities is no more than 400 microns.

[0050] In some embodiments, the longest distance from any location of the biological medium block to the perfusion structure is no more than 200 microns.

[0051] In some embodiments, the perfusion structure forms channels with a diameter ranging from 100-2000 microns.

[0052] In some embodiments, it further comprises the following steps:

[0053] (E) seeding a second culture cell within the perfusion structure, allowing the second culture cell to attach to the inner wall of the perfusion structure.

[0054] In some embodiments, the second culture cell comprises vascular endothelial cells, tumor-associated fibroblasts.

[0055] In some embodiments, the method further comprises the following steps:

[0056] (F) continuously perfusing a culture medium into the perfusion structure, allowing the target cells to proliferate in a three-dimensional manner in the 3D cell culture, resulting in a proliferated cell mass containing target cells.

[0057] In some embodiments, the proliferated cell mass contains a newly formed vascular network structure, and the vascular network structure has one or more of the following characteristics:

[0058] having large blood vessels with a diameter ranging from 10 microns to 100 microns and capillaries with a diameter no more than 10 microns;

[0059] having blood vessels expressing CD31 protein;

[0060] having a plurality of blood vessels interconnected to form a network;

[0061] the vascular network structure is integrated with the target cells.

[0062] In some embodiments, the proliferated cell mass: a) expresses at least one tissue-specific marker of the target tissue from which the target cells are derived.

[0063] In one aspect, the present application provides a 3D cell culture body prepared by the above method.

[0064] In one aspect, the present application provides a 3D cell culture body prepared by the above method.

[0065] a solidified biomedia mass having a three-dimensional network perfusion structure inside the biomedia mass; and

[0066] an encapsulation material covering the outer layer of the solidified biomedia mass;

[0067] wherein, the inner wall of the perfusion structure has a vascular barrier comprising vascular endothelial cells, and the biomedia mass beside the perfusion structure has a cell mass comprising target cells, the cell mass contains a newly formed vascularized network, the vascularized network has one or more of the following characteristics:

[0068] having large vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters not exceeding 10 microns;

[0069] having vessels expressing CD31;

[0070] having a plurality of vessels interconnected to form a network;

[0071] the vascular network structure is fused with the target cells.

[0072] In some embodiments, the cell mass expresses at least one tissue-specific marker of the target tissue from which the target cells are derived.

[0073] In some embodiments, the target cells are lung cancer cells, and the target tissue is lung cancer tissue.

[0074] In some embodiments, the tissue-specific marker comprises a lung cancer marker, a lung cancer extracellular matrix marker, or a vascular marker.

[0075] In some embodiments, the cell culture body satisfies:

[0076] the lung cancer marker comprises one or more markers selected from the group consisting of CK-7 and TTF-1;

[0077] The lung cancer extracellular matrix markers comprise one or more markers selected from the group consisting of FN, Vimentin, COL1A1, MMP2;

[0078] The blood vessel markers comprise CD31.

[0079] In some embodiments, the cell cluster expresses at least simultaneously Vimentin, FN, and CK-7.

[0080] In some embodiments, the markers are detected by immunohistochemistry or immunofluorescence.

[0081] In some embodiments, the lung cancer extracellular matrix markers detected by immunohistochemistry are distributed in the cell membrane.

[0082] In some embodiments, the solidified biomedia block comprises a solidified protein-based gel, a solidified synthetic-based gel, a solidified polysaccharide-based gel, and / or a solidified matrix-based gel.

[0083] In some embodiments, the solidified biomedia block further comprises stromal cells and / or cell inductors.

[0084] In some embodiments, the cell inductors comprise cytokines.

[0085] In some embodiments, the encapsulation material is as defined above.

[0086] In some embodiments, the perfusion structure is formed by a sacrificial material-based method.

[0087] In some embodiments, the perfusion structure can allow perfusion of culture medium to allow the cell cluster to proliferate in a three-dimensional manner in the 3D cell culture body.

[0088] In some embodiments, the 3D cell culture body is capable of allowing at least 1 cell cluster to be cultured to at least 1 cubic centimeter.

[0089] In some embodiments, the 3D cell culture body is capable of allowing at least 1 cell cluster to be cultured for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks.

[0090] In some embodiments, the solidified biomedia block contains at least one cell cluster having a size of at least 10 cubic millimeters and having at least one of the following properties:

[0091] expressing the target tissue molecular markers;

[0092] at least one region contains a newly formed vascularized network having one or more of the following characteristics:

[0093] large blood vessels having a diameter ranging from 10 micrometers to 100 micrometers and capillaries having a diameter of not more than 10 micrometers;

[0094] vessels expressing CD31;

[0095] a plurality of vessels connected to each other to form a network;

[0096] the vessel network structure is fused together with target cells.

[0097] In some embodiments, the size of the cell aggregate is at least 5 cubic millimeters, at least 50 cubic millimeters, at least 100 cubic millimeters, at least 200 cubic millimeters, at least 300 cubic millimeters, at least 500 cubic millimeters, at least 700 cubic millimeters, at least 800 cubic millimeters, at least 900 cubic millimeters, or at least 1 cubic centimeter.

[0098] In some embodiments, the 3D cell culture body is prepared by the preparation method of the present application.

[0099] In one aspect, the present application provides an in vitro proliferated cell aggregate, which is obtained by culturing the above-mentioned 3D cell culture body.

[0100] In one aspect, the present application provides a 3D cell culture device, which has the above-mentioned 3D cell culture body therein.

[0101] In some embodiments, the 3D cell culture device further has a housing for packaging the 3D cell culture body.

[0102] In some embodiments, the 3D cell culture device further has a device capable of applying mechanical force to the 3D cell culture body.

[0103] In some embodiments, the device for applying mechanical force is capable of rotating the 3D cell culture body.

[0104] In some embodiments, the inlet and / or outlet of the perfusion structure is connected to a pump.

[0105] In some embodiments, the pump is controlled by a control system.

[0106] In some embodiments, the inlet and / or outlet of the perfusion structure and the pump are further provided with a device for removing air bubbles.

[0107] In some embodiments, the pump and / or the device for applying mechanical force are controlled by a control system.

[0108] In some embodiments, the 3D cell culture device is a microfluidic device.

[0109] In one aspect, the present application provides a method of testing a candidate drug, comprising:

[0110] (a) providing a 3D cell culture body as described above or a 3D cell culture device as described above;

[0111] (b) perfusing the candidate drug into the perfusion structure of the 3D cell culture body,

[0112] (c) determining the biological activity of the cell mass in the 3D cell culture body after perfusion of the candidate drug to determine the change compared to the biological activity before perfusion, and

[0113] (d) evaluating the effect of the candidate drug on the biological activity of the cell mass based on the change.

[0114] In some embodiments, the cell mass comprises tumor cells.

[0115] In some embodiments, the biological activity of the cell mass comprises proliferation, apoptosis, growth arrest, cell spreading state, cell migration, cell-to-cell connection, and / or cell morphology change of the cell mass.

[0116] In one aspect, the present application provides a method of culturing a tissue, comprising:

[0117] (1) providing a 3D cell culture body as described above or a 3D cell culture device as described above;

[0118] (2) perfusing a culture solution into the perfusion structure of the 3D cell culture body to allow the cell mass to proliferate in three dimensions; and

[0119] (3) obtaining a proliferated cell mass, wherein the proliferated cell mass contains a newly formed vascularized network having one or more of the following characteristics:

[0120] having large blood vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters not exceeding 10 microns;

[0121] having blood vessels expressing CD31;

[0122] having a plurality of blood vessels interconnected to form a network;

[0123] the vascular network structure is fused together with a target cell portion.

[0124] In some embodiments, the method further comprises processing the proliferated cell mass so that it is suitable for being transplanted into a body.

[0125] As used in this application, the terms "about" and "approximately" are used interchangeably. Any numerical values recited in this application, with or without the about / approximately qualifier, are intended to cover any normal fluctuation experienced by a person of ordinary skill in the relevant art.

[0126] Additional features, objects, and advantages of the application are set forth in the detailed description that follows, and will become apparent to those skilled in the art from the detailed description, the claims, and the appended drawings.

[0127] BRIEF DESCRIPTION OF DRAWINGS

[0128] The following drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description, explain the principles of the application.

[0129] Figure 1 is the effect of adjusting the number of secondary culture cells on the culture results;

[0130] Figure 2 is the state of the cells at the third day of culture, wherein A is the green fluorescent labeled lung cancer cell line (A549-GFP), B is the red fluorescent labeled vascular endothelial cells (HUVEC-RFP), and C is the state of the cells under bright field;

[0131] Figure 3 is the state of the lung cancer organoids at the third day of culture (4X), wherein A is the green fluorescent protein labeled organoids, B is the red fluorescent protein labeled blood vessels, C is the superimposed effect diagram, arrow 1 points to the blood vessels, and arrow 2 points to the organoids;

[0132] Figure 4 is the state of the organoid 3D culture body at the third day of culture, and the arrow points to the organoids;

[0133] Figure 5 is the marker expression in the 3D culture body at the third day of culture, wherein A is the lung cancer cell line, B is the lung cancer organoids, CK7 and TTF-1 are lung cancer marker proteins, MMP2, COL1A1 and FN are extracellular matrix marker proteins, Vimentin is a tumor associated fibroblast marker, and CD31 is a vascularization related marker protein;

[0134] Figure 6 is the HE staining diagram of the lung cancer cell line 3D culture body, wherein arrow 1 points to the lung cancer cell line nucleus, arrow 2 points to the cytoplasm, and arrow 3 points to the extracellular matrix;

[0135] Figure 7 is the immunohistochemical staining of the lung cancer cell line 3D culture body, wherein A is FN, B is CD31, C is ck7, and D is Vimentin;

[0136] Figure 8 is a HE staining diagram of lung cancer organoids 3D culture and CD31, FN, ck7, Vimentin immunohistochemical staining. In the HE row, arrow 1 points to the nucleus, arrow 2 points to the cytoplasm, and arrow 3 points to the extracellular matrix. The scale of the HE diagram in the Day 1 column is 100 μm. The scales of the CD31, CK7, FN, and Vimentin diagrams are 50 μm. In the Day 3 column, the scales of the CD31 and CK7 diagrams are 100 μm, and the scales of the FN and Vimentin diagrams are 50 μm. In the tissue column, the scales of the HE and CK7 diagrams are 200 μm.

[0137] Figure 9 is an enlarged HE staining result. Arrow 1 points to a blood vessel, arrow 2 points to a blood vessel endothelial cell, and arrow 3 points to tumor tissue or tumor nest tissue.

[0138] Figure 10 shows the implementation process of one specific embodiment of the method of the present application, in which A is a top view of a printed skeleton structure, B is the content of the gel added in the process, and C is the complete manufacturing process.

[0139] Figure 11 shows a microfluidic chip for printing a skeleton, in which 1 is a magnet for sealing, 2 is a liquid inlet / outlet for infusion, 3 is a region for printing a skeleton and also a region for culturing cells, 4 is an upper cover, and 5 is a base.

[0140] [Rule 91, 27.06.2025] Figure 12 is a top view of a microfluidic chip for printing a skeleton and its main dimensions, in which 1 represents a magnet for sealing, 2 represents a liquid inlet / outlet for infusion, and 3 represents a region for printing a skeleton and also a region for culturing cells.

[0141] [Rule 91, 27.06.2025] Figure 13 is a front view of a microfluidic chip for printing a skeleton and its main dimensions, in which 4 represents an upper cover and 5 represents a base.

[0142] [Rule 91, 27.06.2025] Figure 14 is a left view of a microfluidic chip for printing a skeleton and its main dimensions.

[0143] [Rule 91, 27.06.2025] Figure 15 is a structural diagram of a microfluidic chip for printing a skeleton.

[0144] [Rule 91, 27.06.2025] Figure 16 shows the application of a chip device in an anti-vascular drug test, in which A is a negative control, B is a positive control, C is a 300 ng / mL concentration drug group, D is a 33 mg / mL concentration drug group, E is a 3.7 ng / mL concentration drug group, and F is the inhibition rate statistical result of different concentration drug groups.

[0145] Definitions

[0146] To facilitate an understanding of the present application, a number of terms are defined below. Additional definitions for the following terms and other terms can be found throughout the specification.

[0147] About or approximately: As used herein, the term "about" or "approximately," when applied to one or more values of interest, refers to a value that is similar to a specified reference value. In certain embodiments, unless otherwise stated, the term "about" or "approximately" means 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 the stated reference value in either direction (greater than or less than) unless otherwise stated or otherwise apparent from the context.

[0148] Unless otherwise expressly stated, in the specification and claims, the term "comprising" or variations such as "comprise" or "comprises" will be understood to mean the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements.

[0149] DETAILED DESCRIPTION

[0150] The specific embodiments of the present application will now be described in detail below. It should be appreciated that the application protects scope is not limited to the specific embodiments.

[0151] Various aspects of the present application are described in further detail in the following subsections. The use of subsections is not intended to limit the application. Each subsection can be applied to any aspect of the application. In this application, unless otherwise stated, the use of "or" means "and / or."

[0152] The present application provides a method capable of easily simulating vascularized structure and simulating microenvironment, which can realize vascularization of organoids and other 3D culture bodies, and can realize long-term and realistic culture of organoids and other 3D culture bodies through the control of medium flow by microfluidic technology.

[0153] To achieve the above object, the technical scheme provided by the present application is as follows:

[0154] I. Preparation method of 3D cell culture body

[0155] In one aspect, the present application provides a method for preparing a 3D cell culture, comprising the following steps: (A) constructing a scaffold based on a sacrificial material; (B) disposing a first culture cell and a solidifiable biomedia around the scaffold, and then solidifying the biomedia to form a solidified biomedia block; (C) covering the biomedia block with a solidifiable encapsulation material, and then solidifying the encapsulation material to form an encapsulation layer outside the biomedia block; (D) removing the scaffold, thereby forming a perfusion structure inside the biomedia block to obtain the 3D cell culture. In some embodiments, the steps are performed in the order of steps (A), (B), (C), and (D).

[0156] sacrificial material

[0157] In step (A), a scaffold based on a sacrificial material is constructed. As used herein, the term "sacrificial material" refers to a material that is used to form a scaffold for forming a perfusable structure, and by removing the scaffold, a cavity as a perfusable structure can be formed. In some embodiments, the sacrificial material has the property of being able to convert between a solid state and a flowable state. In some embodiments, the sacrificial 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 sacrificial material is preferably not biologically toxic. In some embodiments, the sacrificial material is a temperature-sensitive material. In some embodiments, the conditions for converting the sacrificial material to a flowable state have no significant effect on the cultured cells. The sacrificial material comprises Pluronic F-127 and / or gelatin. In some embodiments, the sacrificial material comprises Pluronic F-127 at a mass concentration of 30%-50%, or 30%-40% (w / v), for example, Pluronic F-127 at a mass concentration of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% (w / v). In some embodiments, the sacrificial material comprises gelatin at a mass concentration of 5%-15%, or 10%-15% (w / v), for example, gelatin at a mass concentration of 10%, 11%, 12%, 13%, 14%, 15% (w / v). In some embodiments, the sacrificial material comprises Pluronic F-127 at a mass concentration of 30%-50%, or 30%-40% (w / v), for example, Pluronic F-127 at a mass concentration of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% (w / v) and / or gelatin at a mass concentration of 5%-15%, or 10%-15% (w / v), for example, gelatin at a mass concentration of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% (w / v). In some preferred embodiments, the sacrificial material comprises Pluronic F-127 at a mass concentration of 40% (w / v) and / or gelatin at a mass concentration of 10% (w / v), at this time, the sacrificial material converts between a liquid state and a solid state with temperature change, so that the temperature can be changed to change it from a solid state to a liquid state at a suitable time, facilitating removal, and more importantly, both Pluronic F-127 and gelatin are not biologically toxic, and at the same time, the rate of morphological conversion is fast, and the temperature change range will not affect the cells.

[0158] Scaffold for forming a perfusable structure

[0159] In some embodiments, the scaffold for forming the perfusion structure can have any shape. In some embodiments, the scaffold for forming the perfusion structure preferably has a three-dimensional network structure. In some embodiments, the scaffold for forming the perfusion structure can be formed by any method. In some embodiments, at least a portion of the scaffold for forming the perfusion structure is constructed by 3D printing. In some embodiments, the scaffold for forming the perfusion structure is formed by 3D printing. In some embodiments, the diameter of the sacrificial material linear structure (i.e., one-dimensional structure) constituting the three-dimensional network structure is in the range of 100-2000 microns, optionally in the range of 200-900 microns, 300-800 microns, 400-600 microns, to mimic the diameter of small blood vessels in the human body. In some embodiments, the center-to-center distance (i.e., line distance) between adjacent sacrificial material linear structures constituting the three-dimensional network structure is in the range of 1000-2000 microns, optionally in the range of 1100-1400 microns, 1200-1300 microns, to ensure sufficient material exchange distance. In some embodiments, the overall appearance of the three-dimensional network structure formed can be any desired three-dimensional shape, such as, but not limited to, a cylinder, a cuboid, or a hexahedron. In some preferred embodiments, the 3D printer used in the 3D printing has an extrusion printing function. In some embodiments, the 3D printer has a temperature control module with a controllable temperature range of 0-40°C. In some embodiments, the extrusion needle of the 3D printer is of a model with a diameter in the range of 500-1000 microns. In some embodiments, the 3D printer has a software module that can control the line distance and layer height. In some embodiments, the printing nozzle of the 3D printer is pneumatically controlled. In some specific embodiments, the 3D printing uses one or more of the following printing conditions: the line distance is controlled to be in the range of 1-1.5 mm; the layer height is 0.8 times the diameter of the selected needle; the printing speed is controlled to be in the range of 2-3 mm / s; the pneumatic control air pressure is controlled to be in the range of 0.1-0.2 MPa; and the printing temperature is set to be in the range of 10-25°C.

[0160] Biological medium and first culture cells

[0161] In step (B), first cultured cells and a solidifiable biomedia are disposed around the scaffold, and the biomedia is solidified to form a solidified biomedia block. As used herein, the term "first cultured cells" refers to the cells disposed in step (B). In some embodiments, the first cultured cells comprise target cells, which 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, a cell pellet, etc. containing the target cells. For example, the first cultured cells can be provided in the form of an organoid. As used herein, the term "organoid" refers to a tissue-like construct with a certain spatial structure formed by in vitro three-dimensional (3D) culture of 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. Currently, intestinal organoids, salivary gland organoids, etc. have entered clinical trials, and the use of stomach organoids, pancreatic organoids, thyroid organoids, liver organoids has also been verified.

[0162] The target cells can be obtained by methods well known to the skilled person, for example, by collecting from an organism and / or by differentiating cells having differentiation ability. 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.

[0163] In some embodiments, the target cells are normal cells and / or disease state cells. In some embodiments the target cells can comprise tissue cells of any organ. In some embodiments, the target cells comprise one or more selected from the group consisting of: cardiac tissue cells, brain tissue cells, intestinal tissue cells, stomach 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, breast tissue. As used herein, the term "XX (organ name) tissue cells" refers to a population of cells having the same or substantially the same biological characteristics as the population of cells that make up the particular organ, such a population of cells can be obtained, for example, by harvesting from an organism and / or by differentiating cells having the ability to differentiate. In some embodiments, the disease state cells are tumor cells. In some embodiments, the target cells can comprise any particular kind of cell. 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 combinations thereof. In some embodiments, the target cells comprise tumor cells. In some embodiments, the tumor cells are derived from: lung cancer, breast cancer, liver cancer, intestinal cancer, stomach cancer, kidney cancer, and the like.

[0164] In some embodiments, the first culture cells further comprise other cells for improving the culture environment of the target tissue cells, such as stromal cells, etc. By adding these auxiliary cells, the complex environment surrounding the target tissue can be further simulated, and the mixture of multiple cells can more highly simulate the in vivo environment, and the biological factors released by the cells to each other also have a positive effect on their morphology. In some embodiments, the first culture cells further comprise one or more cells selected from the group consisting of stromal cells, vascular-like cells. In some embodiments, the stromal cells are selected from fibroblasts, or mesenchymal stem cells. In some embodiments, the stromal cells and / or vascular-like cells can have the same or different origins 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 proportion of stromal cells in the first culture cells is 10%-90%, preferably 20%-70%, more preferably 30%-50%. In some embodiments, the ratio of stromal cells to target cells is: 1:3-10:1; preferably: 1:3-5:1; more preferably 1:2-3:1, more preferably 3:1. In some embodiments, the vascular-like cells and the target cells are derived from the same species. In some embodiments, the vascular-like cells are selected from vascular endothelial cells or vascular-like organs. In some embodiments, the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC cells). In some embodiments, the proportion of vascular-like cells in the first culture cells is 10%-90%, preferably 20%-70%, more preferably 30%-50%. In some embodiments, the ratio of vascular-like cells to target cells is: 1:1-10:1; preferably: 1:1-5:1; more preferably 1:1-3:1.

[0165] In some embodiments, the first culture cells and the solidifiable biomedia are arranged around the skeleton. In some embodiments, the first culture cells are dispersed in the biomedia.

[0166] 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 photo-curing, thermal curing, vibration-induced crosslinking, etc. In some embodiments, the biological medium has lower biotoxicity than the encapsulating material. In some preferred embodiments, the biological medium has no biotoxicity. In some embodiments, the biological medium is biodegradable, whereby the extracellular matrix of the cultured cells can replace the original biological medium after a long period of culture, further mimicking the cell microenvironment in a natural environment. In some embodiments, the curing condition of the biological medium has no significant effect on the cultured cells.

[0167] As used herein, the term "biotoxicity" refers to the adverse effect of a material on the life activities of biological cells, such as growth, differentiation, lifespan, etc. In some embodiments, biotoxicity refers to cytotoxicity, i.e., the adverse effect caused by affecting the basic structure and / or physiological processes of the cultured cells, leading to disorder of cell survival, proliferation and / or function. Biotoxicity or cytotoxicity can be tested by any means known in the art.

[0168] 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-based gel, a synthetic-based gel, a polysaccharide-based gel, and / or a matrix-based gel. In some embodiments, the protein-based gel comprises one or more selected from the group consisting of collagen I, fibrin, gelatin, elastin, fibroin, or a combination thereof. In some embodiments, the synthetic-based gel comprises one or more selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-isopropyl acrylamide) (PNIPAM), polyethylene oxide (PEO), polyglycolic acid (PGA), or a combination thereof. In some embodiments, the polysaccharide-based gel comprises one or more selected from the group consisting of hyaluronic acid, alginate, chitosan, dextran, chondroitin sulfate. In some embodiments, the matrix-based gel comprises one or more selected from the group consisting of Matrigel TM , MaxGel TM , CD-ECM, Tissue-specific dECM Hydrogel, etc.

[0169] 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 that has the ability to modulate the growth, differentiation, and / or effect of a cell. As used herein, the term "cytokine" refers to a secreted polypeptide or secreted protein that has the ability to modulate the growth, differentiation, and / or effect of a cell, generally having a small 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 the group consisting of Retinoic acid (RA), TWS119, Cardiogenol A-D, IDE1, and IDE2. In some embodiments, the cytokine is selected from the group consisting of EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Wnt-3a. In a specific embodiment, the first culture cells comprise lung cancer cells, and the cytokine is selected from the group consisting of EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Wnt-3a, Y-27632. Different kinds of cytokines are selected to have an impact on the growth state of the organoids, and the present application has screened and determined the above cytokines that are beneficial for cell growth and differentiation.

[0170] In some embodiments, the first culture cells and the solidifiable biological medium can be disposed around the scaffold by any technical method known to the skilled person. Including but not limited to: injection using a pipette tip or the like, 3D printing, coating, perfusion around the scaffold, etc. In some embodiments, the disposing is performed by applying the biological medium with the first culture cells dispersed therein on the scaffold. In some embodiments, the disposing can be performed by applying the biological medium with the first culture cells dispersed therein on the scaffold (or previously disposed and solidified biological medium). In some embodiments, the applying can be performed by coating or the like. In some embodiments, the disposing can be performed by perfusing the biological medium with the first culture cells dispersed therein around the scaffold.

[0171] In some embodiments, the operation of disposing the first cultured cells and the solidifiable biological medium around the skeleton can be performed in one or more times, and the biological medium, the first cultured cells and / or the disposing method used in each disposing operation can be the same or different from each other. A specific embodiment will be described in detail below, and it should be noted that the following examples are only used to help understanding and do not have any intention to limit the specific embodiments or the protection scope of the present application. For example, the first cultured cells and the solidifiable biological medium can be disposed in 3 times, and in this case, the biological medium A can be disposed first and solidified, then the biological medium B is disposed and solidified, and finally the biological medium C is disposed and solidified, where A, B and C are only used to indicate the order of the biological medium disposing and do not have any intention to indicate that the three are the same or different in composition. Among them, the biological medium A, the biological medium B and the biological medium C can be the same or different from each other. Among them, one or two of the biological medium A, the biological medium B and the biological medium C can not contain the first cultured cells (at least one biological medium contains the first cultured cells). Among them, the biological medium A, the biological medium B and the biological medium C can contain the same or different first cultured cells to achieve the spatial disposition between different first cultured cells. Among them, the disposing method between multiple disposing operations can be the same or different, for example, in the case of disposing the biological medium A and / or the biological medium B by coating, the biological medium C can be disposed by perfusion.

[0172] Encapsulating material

[0173] In step (C), the biomedia block is covered with a curable encapsulating material, and then the encapsulating material is cured to form an encapsulating layer on the outside of the biomedia block. As used herein, the term "encapsulating material" refers to a material used to encapsulate the outermost layer of the biomedia to provide support and reinforcement to the 3D culture and to prevent leakage, and through the encapsulation and reinforcement by the encapsulating material, the 3D culture of the present application can be continuously cultured for a longer period without breakage and loss during perfusion culture. In some embodiments, the encapsulating material has a higher strength after curing than the biomedia. The strength of a material can be described and detected in a manner conventional in the art. It is noted that the strength used herein refers to the ability of a material to maintain its shape under perfusion culture, rather than the strict mechanical strength. In some embodiments, the encapsulating material has any one or more of the following characteristics: 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 a loss modulus of 2,000-50,000 Pa, preferably 5,000-45,000 Pa, more preferably 10,000-40,000 Pa, more preferably 20,000-30,000 Pa. In some embodiments, the encapsulating material has a higher strength after curing than the biomedia refers to that the storage modulus and / or loss modulus of the biomedia is lower than that of the encapsulating material. In some embodiments, the storage modulus and / or loss modulus can be detected in a manner conventional in the art. In some embodiments, the storage modulus and / or loss modulus are detected by a rheometer. In some embodiments, the encapsulating material is a curable material, and the curing mechanism can be any one known in the art, such as photocuring, thermal curing, vibration-induced crosslinking, etc. Preferably, the encapsulating material is not biologically toxic. In some embodiments, the encapsulating material is biodegradable. In some embodiments, the curing condition of the encapsulating material has no significant effect on the cultured cells. In some embodiments, the encapsulating material comprises one or more selected from the group consisting of a heat-sensitive material, a light-sensitive material, or a combination thereof. In some embodiments, the light-sensitive material is a photocured gel.In some embodiments, the photocured gel comprises one or more of methacrylated collagen (COMA), methacrylated chitosan (CSMA), methacrylated silk fibroin (SILMA), methacrylated gelatin (GelMA), hyaluronic acid hydrogel (HAMA), methacrylated dextran (DexMA), methacrylated sodium alginate (AlgMA), or a combination thereof. In some embodiments, the biological medium is collagen I or Matrigel. TM MaxGel TM CD-ECM, Tissue-specific dECM Hydrogel, etc., and the encapsulation material is GelMA. In some embodiments, the encapsulation material has a higher strength after curing than the biological medium after curing; and optionally, the biological medium and / or the encapsulation material is biodegradable. In some embodiments, the biological medium is collagen I or Matrigel, and the encapsulation material is GelMA.

[0174] Perfusion structure after removal of sacrificial material

[0175] In step (D), the scaffold is removed, thereby forming a perfusion structure inside the biological medium block, to obtain the 3D cell culture. As used herein, the term "perfusion structure" refers to the cavity structure formed in the cured biological medium block after the removal of the scaffold. In some embodiments, the perfusion structure is designed to have one or more inlets and / or outlets for perfusion, it is noted that the inlets and outlets herein can be interchangeable depending on the specific use. As described in the section of sacrificial material, the conditions for removing the sacrificial material preferably have no significant effect on the cultured cells. For example, when the sacrificial material is a temperature-sensitive material, the step (D) comprises removing the scaffold at a temperature ranging from 0-37 °C, optionally, the specific temperature is 0-10 °C, preferably 3-6 °C, more preferably 4 °C. In a preferred embodiment, when the sacrificial material is a temperature-sensitive material, the process of removing the above-mentioned sacrificial material should be controlled within 30 minutes to avoid significant effects on the cultured cells. It is noted that the selection of the sacrificial material and the biological medium should meet the condition that the curing condition of the biological medium and the removal condition of the sacrificial material should not be the same or similar, to avoid the sacrificial material becoming flowable during the curing process of the biological medium.

[0176] The perfusion structure can have any structure, and the skilled person can design it as needed to achieve the perfusion effect. In some embodiments, the perfusion structure has a three-dimensional network-like pipe cavity. In some embodiments, the maximum distance between adjacent pipes in the three-dimensional network-like pipe cavity is not more than 400 microns. In some embodiments, the shortest distance from any position of the biological medium block to the perfusion structure is not more than 200 microns. In some embodiments, the diameter of the pipe formed by the perfusion structure is between 500-1000 microns, optionally between 600-900 microns, or between 700-800 microns. In some embodiments, the diameter of the pipe formed by the perfusion structure is between 500-1000 microns, optionally between 600-900 microns, or between 700-800 microns.

[0177] Second culture cells

[0178] In some embodiments, second culture cells can be optionally further configured on the inner wall of the perfusion structure, thereby simulating the required tissue structure, such as the vascular wall structure, on the inner wall of the perfusion structure. That is, in some embodiments, the preparation method of the 3D cell culture body further comprises the following step: (E) seeding second culture cells in the perfusion structure, so that the second culture cells adhere to the inner wall of the perfusion structure. As used herein, the term "second culture cells" refers to cells introduced into the perfusion structure through the inlet of the perfusion structure, only for the purpose of distinguishing the configuration from the first culture cells, without any indication that the first culture cells and the second culture cells are the same or different in composition and other aspects.

[0179] In some embodiments, by configuring appropriate second culture cells, the vascular structure can be further simulated. For example, in some embodiments, the second culture cells comprise vascular endothelial cells. In some embodiments, the second culture cells further comprise 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, and more preferably 1:1. When the culture system only contains cancer-associated fibroblasts or vascular endothelial cells, subsequent culture cannot form blood vessels. In some embodiments, the vascular endothelial cells are provided in the form of vascular organoids or vascular spheroids. In some embodiments, the seeding is carried out in a manner such that the final density of the vascular endothelial cells is in the order of 1x10 7 6 6 1.0x102.0x103.0x106 One, approximately 4.0 × 10 6 Each, approximately 5.0 × 10 6 Each, approximately 6.0 × 10 6 Each, approximately 7.0 × 10 6 One, approximately 8.0 × 10 6 One, approximately 9.0 × 10 6 Seeding is carried out at a density of [number] cells. In some embodiments, the final density of the vascular endothelial cells is 3.0 × 10⁶ cells / year. 6 Seeding is performed at a rate of 1 cell / mL. Insufficient cell density leads to uneven cell adhesion, hindering the formation of a vascular barrier and affecting subsequent germination and the formation of capillary-like structures, as shown in Figure 1 (cell density 5.0 × 10⁻⁶). 5 However, excessively high cell density leads to a significant waste of cells and also presents problems in terms of 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 to the culture vessel.

[0180] In and / or after step (E), in order to promote the adhesion of the second cultured cells to the inner wall of the perfusion structure, techniques known in the art may be used, such as adding cell inducers and / or cytokines to the perfusion fluid containing the second cultured cells; applying mechanical force, etc.

[0181] In some embodiments, techniques in the art can be further employed to promote the growth of vascular endothelial cells, thereby forming a vascular barrier to mimic 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 method for preparing the 3D cell culture further includes the following steps: (F) perfusing culture medium into the perfusion structure 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), techniques known in the art can be used to generate the vascular barrier, such as adding cell inducers and / or cytokines (e.g., VEGFA, FGF10, FGF7, Noggin, RSPO1, and Wnt-3a, etc.) to the culture medium; applying mechanical force, etc. Under the porous structure of the biomedia block, the vascular barrier will invade and bud into the biomedia block, further forming newly formed vascular-like structures, eventually intertwining with cell clusters formed by the proliferation of target cells, thereby mimicking the real vascularized microenvironment of tissues in vivo.

[0182] Target cell culture

[0183] In some embodiments, the method of preparing the 3D cell culture further comprises the following step: (G) continuously perfusing 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 proliferated cell mass containing target cells. In some embodiments, the medium can be perfused at any suitable speed. In some embodiments, the perfusion speed is 1-20 μL / min, preferably 3-15 μL / min; more preferably 5-10 μL / min. Too fast a speed can cause the medium to disperse throughout the vascularized structure, and too slow a speed can not provide an effective shear force on the cells, affecting the formation of blood vessels. As used herein, the term "cell mass" refers to a cell colony that has proliferated in a three-dimensional manner. The cell mass can be formed by the proliferation of one cell colony, or can be formed by the fusion of multiple cell colonies after proliferation.

[0184] The present application surprisingly found that by performing the above steps (A) to (G), not only a 3D cell culture with perfusion structure can be obtained, but also the newly formed blood vessel-like structure on the blood vessel barrier can be fused and intertwined with the cell mass, forming an ideal structure similar to the real vascularized microenvironment. Such structure is similar to the distribution pattern of natural capillary blood vessels, which can further improve the continuous culture ability of the 3D cell culture, which has not been obtained in the prior art. That is, in some embodiments, the proliferated cell mass contains a newly formed blood vessel network structure, which has one or more of the following characteristics: i) has large blood vessels with a diameter ranging from 10 microns to 100 microns and capillaries with a diameter of no more than 10 microns; ii) has blood vessels expressing CD31; iii) has multiple blood vessels connected to form a network; and iv) the blood vessel network structure is partially fused with the target cells. In some embodiments, the blood vessel network structure further has the following characteristics: v) the blood vessel network structure allows perfusion culture; in some embodiments, the blood vessel network structure is fused with the target cells. In some embodiments, the blood vessel network structure is connected to the perfusion structure. As used herein, the term "newly formed" refers to structures formed after perfusion culture, not original structures in the culture material. As used herein, the term "fusion" refers to the close connection of the tip cells after budding in the blood vessel network structure with the target cells, or the close connection of the blood vessel-like structure and other cells to form a multi-layer cell tissue-like structure. As used herein, the term "capillary" refers to a blood vessel-like structure with a diameter of no more than 10 microns. As used herein, the term "large blood vessel" refers to a blood vessel-like structure with a diameter of 10 microns to 100 microns, relative to capillaries. In some embodiments, large blood vessels include blood vessel-like structures with a diameter of 10 microns to 20 microns, a diameter of 20 microns to 30 microns, a diameter of 30 microns to 40 microns, a diameter of 40 microns to 50 microns, a diameter of 50 microns to 60 microns, a diameter of 60 microns to 70 microns, a diameter of 70 microns to 80 microns, and / or a diameter of 80 microns to 100 microns.

[0185] Similarity to target tissue

[0186] The inventors of the present application have also surprisingly found that, compared to the prior art, the cell aggregates cultured in the 3D cell culture body of the present application have more similar biological characteristics to the target tissue in the natural state. As used herein, the term "target tissue" refers to the tissue in the natural state that the target cells are expected to mimic. For example, the target tissue of a liver organoid is liver, and for another example, the target tissue of "XX (organ name) tissue cells" is XX (organ name) tissue. Without being bound by any theory, the inventors of the present application speculate that this is because the blood vessel-like structure of the present application, the ideal structure of the blood vessel-like structure and the cell aggregate, can more closely mimic the natural blood vessel 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 proliferated cell aggregate: a) expresses at least one target tissue molecular marker that is specifically expressed in the target tissue of the target cells; and / or b) has a gene sequencing result similar to the target tissue. In some embodiments, the target tissue molecular markers include extracellular matrix markers of the target tissue, markers of the target cells of the target tissue, epithelial-mesenchymal transition markers, and / or vascular markers.

[0187] In some embodiments, the target cells are lung cancer cells, 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, 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 group consisting of FN, COL1A1, MMP2, Vimentin; c) the vascular markers include CD31. In one embodiment, the cell aggregate expresses lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition markers, and vascular markers simultaneously. In one embodiment, the cell aggregate expresses at least FN and Vimentin simultaneously. The expression of the markers can be detected by methods known in the art. For example, by immunohistochemistry or Western Blot.

[0188] Advantages of the present application

[0189] The method provided by the application can construct a multi-layer space vascularized organoid, can vascularize around the cultured cells or tissues, and thus can obtain nutrient factors without obstacles and discharge metabolic waste in time, thereby simulating the in-vivo microenvironment. The previous vascularized gel structure is difficult to realize the internal space and culture mode of the arbitrary structure. In addition, an important content is that the application can use a dynamic culture mode, which is different from the traditional completely closed culture. The completely closed culture is difficult, and in many existing models, it is difficult to realize long-term culture. The application can also input the interaction between immune cells and tissues, and the constructed tissue model can break through the limitation of the traditional chip and maximize the size.

[0190] The application utilizes a sacrificial material to form a pipe structure, co-prints with a printing ink containing a biological medium, finally integrates a thin-wall tissue, interstitial tissue and cultured cells into a single thick tissue block, and then lays a vascular endothelial cell lining in the perfusable pipe structure and forms a barrier. The thick tissue block can contain growth factors, induce the sprouting of endothelial cells and optional fibroblasts, and finally combine the formed capillary network and the cultured cell mass in the tissue block to form a perfusable vascularized cell mass in the microenvironment. For example, the application prints a skeleton based on a sacrificial material and uses it as a cell-compatible sacrificial template to generate a network of tubular cavities in an engineered tissue containing living cells, which can be lined with endothelial cells and perfused with a culture solution under high pulsatile flow. Since this simple blood vessel casting method can independently control the network geometry, endothelialization and extravascular tissue, it is compatible with various cell types, synthetic and natural extracellular matrices, and crosslinking schemes.

[0191] II. 3D cell culture body

[0192] In one aspect, the present application provides a 3D cell culture body prepared by the 3D cell culture body preparation method of the present application.

[0193] In another aspect, the present application provides an in-vitro cultured 3D cell culture body comprising: a) a solidified biological medium block, and having a stereoscopic network perfusion structure inside the biological medium block; and b) an encapsulation material covering the outer layer of the solidified biological medium block; wherein the inner wall of the perfusion structure has a vascular barrier comprising vascular endothelial cells, and the biological medium block beside the perfusion structure has a cell mass comprising target cells, and the cell mass contains a newly formed blood vessel-like structure. In some embodiments, the 3D cell culture body can be prepared by or not by the preparation method of the 3D cell culture body.

[0194] In some embodiments, the 3D cell culture contains a nascent vascular network structure, which has one or more of the following characteristics: i) has large vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters no more than 10 microns; ii) has vessels expressing CD31; iii) has multiple vessels interconnected to form a network, and v) the vascular network structure is integrated with the target cells. In some embodiments, the vascular network structure is integrated with the target cells. As used herein, the term "integrated" refers to the close association of the vascular endothelial cells in the vascular network structure that form the vessel-like structures with the target cells. As used herein, the term "capillary" refers to a vessel-like structure with a diameter no more than 10 microns. As used herein, the term "large vessel" refers to a vessel-like structure with a diameter of 10 microns to 100 microns, in relation to a capillary. In some embodiments, the large vessels comprise vessel-like structures with diameters of 10 microns to 20 microns, vessel-like structures with diameters of 20 microns to 30 microns, vessel-like structures with diameters of 30 microns to 40 microns, vessel-like structures with diameters of 40 microns to 50 microns, vessel-like structures with diameters of 50 microns to 60 microns, vessel-like structures with diameters of 60 microns to 70 microns, vessel-like structures with diameters of 70 microns to 80 microns, and / or vessel-like structures with diameters of 80 microns to 100 microns.

[0195] In some embodiments, the cell aggregates in the 3D cell culture have similarity to the target tissue, and the description of "similarity" can refer to the relevant recitations in the section "similarity to the target tissue". In particular, in some embodiments, the cell aggregates express at least one target tissue molecular marker that is specifically expressed in the target tissue of origin of the target cells. In some embodiments, the target tissue molecular marker comprises an extracellular matrix marker of the target tissue, a marker of the target cells of the target tissue, and / or a vascular marker.

[0196] In some embodiments, the target cells are lung cancer cells, and the target tissue is lung cancer tissue. In some embodiments, the molecular markers of the target tissue include lung cancer markers, lung cancer extracellular matrix markers, epithelial-mesenchymal transition (EMT) markers, and / or vascular markers. In some embodiments, the molecular markers of the target tissue include lung cancer markers, lung cancer extracellular matrix markers, EMT markers, and / or vascular markers. In some embodiments, 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 group consisting of FN, COL1A1, and MMP2Vimentin; c) the vascular markers include CD31. In one embodiment, the cell clump simultaneously expresses lung cancer markers, lung cancer extracellular matrix markers, EMT markers, and vascular markers. In one embodiment, the cell clump simultaneously expresses at least FN and Vimentin. The expression of the markers can be performed using methods known in the art. For example, it can be detected using immunohistochemistry or Western blotting.

[0197] The concepts of "biological medium" and "cured biological medium block" can be found in the relevant description in the section "Preparation Method of 3D Cell Culture". Specifically, in some embodiments, the cured biological medium block comprises a cured protein gel, a cured synthetic gel, and / or a cured matrix gel. In some embodiments, the biological medium further comprises a cell inducer. In some embodiments, the cell inducer comprises cytokines. In some embodiments, the concept of "encapsulation material" can be found in the relevant description in the section "Encapsulation Material". The concept of "perfusion structure" can be found in the relevant description in the section "Perfusion Structure". The concept of "sacrificial material" can be found in the relevant description in the section "Sacrificial Material". Specifically, in some embodiments, the perfusion structure is formed by a method based on 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. 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 are interchangeable depending on the specific use.

[0198] The inventors of this invention unexpectedly discovered that the 3D cell culture of this invention can overcome the size limitations and continuous culture duration limitations in 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 more than 12 weeks. Without being limited by any theory, the inventors speculate that this is because the vascular-like structure of this invention, and the ideal structure of the vascular-like structure fused with the cell cluster, can more closely resemble the natural vascular structure and have a material exchange capacity close to that of the natural vascular structure. That is, in some preferred embodiments, the 3D cell culture has the following characteristics: it contains at least one cell cluster with a size of at least 10 cubic millimeters in the solidified biological medium block and has at least one of the following properties: a) expressing the target tissue molecular marker; b) at least one region containing a capillary-like structure with a diameter not exceeding 10 micrometers. In some preferred embodiments, the size of the cell clusters is at least 20 cubic millimeters, at least 50 cubic millimeters, at least 100 cubic millimeters, at least 200 cubic millimeters, at least 300 cubic millimeters, at least 500 cubic millimeters, at least 700 cubic millimeters, at least 800 cubic millimeters, at least 900 cubic millimeters, or at least 1 cubic centimeter.

[0199] III. Cell clusters proliferated in vitro

[0200] In one aspect, the present invention provides an in vitro proliferating cell mass obtained by culturing the 3D cell culture medium of the present invention. In one embodiment, at least one region of the cell mass contains a newly formed vascular network structure having one or more of the following characteristics: i) having large blood vessels with a diameter ranging from 10 micrometers to 100 micrometers and capillaries with a diameter not exceeding 10 micrometers; ii) having blood vessels expressing CD31; iii) having 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.

[0201] IV. Culture Apparatus

[0202] The present invention also provides a culture apparatus for continuously culturing the 3D cell culture. Specifically, in one aspect, the present invention provides a 3D cell culture apparatus having the 3D cell culture of the present invention. In some embodiments, the 3D cell culture apparatus further includes a shell for packaging the 3D cell culture. The shell can be made of any material, preferably a non-biotoxic material. In some embodiments, to provide the mechanical force required for cell culture, the 3D cell culture apparatus further includes 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 various tests described below, including, but not limited to, rotation, vibration, and shaking. That is, in some embodiments, the device for applying mechanical force is capable of rotating the 3D cell culture. In some embodiments, the culture apparatus for the 3D cell culture has a pump for infusing liquid into a perfusion structure. In some embodiments, the inlet and / or outlet of the perfusion structure is connected to the pump. The term "pump" refers to any device used to transport or pressurize fluid. It should be noted that, due to factors such as the action of a pump, the perfusion fluid may contain air bubbles. These air bubbles, due to shear forces and other factors when they burst, may adversely affect the cultured cells. Therefore, in some preferred embodiments, a device for removing air bubbles is further provided between the inlet and / or outlet of the perfusion structure and the pump. The device for removing air bubbles is not particularly limited, as long as it can eliminate air bubbles from the fluid.

[0203] In one aspect, the culture apparatus of the present invention is capable of controlled 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 apparatus to achieve controlled cell culture. In some embodiments, the control system includes a computer. In some embodiments, the pump and / or the device for applying mechanical force is controlled by the control system. In some embodiments, the 3D cell culture apparatus 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 via microchannels.

[0204] V. Uses

[0205] The 3D cell culture body and / or culture device of the present invention can be used efficiently for three-dimensional cell culture, such as for 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.

[0206] The 3D cell cultures of this 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 have the potential to predict individual patient responses to treatment. For example, a live biobank of tumor organoids from patients with metastatic gastrointestinal cancer has reproduced these patients' responses to anticancer drugs in clinical trials. Tumor organoids can also be used to study tumor niches. Organoid libraries representing different grades of colorectal tumors have revealed a decrease in dependence on niche factors during the transition from normal tissue to adenoma to cancer; it was found that niche factor dependence is mainly related to the tumor's genomic composition. 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. Furthermore, organ toxicity is a major cause of drug development failure and post-approval withdrawal. Current toxicology screening using cell lines and animal models often fails to predict adverse reactions in humans (of which renal and hepatic 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 kidney progenitor cells have a near-unlimited self-renewal capacity in 3D culture, which could be a boon for the standardization of nephrotoxicity screening. The U.S. Food and Drug Administration (FDA) has begun using three-dimensional "liver-on-a-chip" organoid models to detect hepatotoxicity in food additives, nutritional supplements, and cosmetics. (Li, Mo, and Juan C. Izpisua Belmonte. "Organoids—Preclinical Models of Human Diseases." New England Journal of Medicine 380.6(2019):569-579.)

[0207] 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 clumps in the 3D cell culture after perfusion of the candidate drug to determine changes in bioactivity compared to pre-perfusion levels; and d) evaluating the effect of the candidate drug on the bioactivity of the cell clumps based on the changes. In some embodiments, the bioactivity of the cell clumps includes, but is not limited to, cell proliferation, apoptosis, growth arrest, cell unfolding state, cell migration, cell-cell connections, and / or changes in cell morphology. In some embodiments, the method is used for screening antitumor drugs. In some embodiments, the cell clumps contain tumor cells.

[0208] 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 culture medium into the perfusion structure of the 3D cell culture body to allow the cell clumps to proliferate in a three-dimensional manner; and c) obtaining the proliferated cell clumps, wherein the proliferated cell clumps contain vascular-like structures. In some embodiments, at least one region of the proliferated cell clumps contains a newly formed vascular network structure having one or more of the following characteristics: having large blood vessels with a diameter ranging from 10 micrometers to 100 micrometers and capillaries with a diameter not exceeding 10 micrometers; having blood vessels expressing CD31; and the vascular network structure being 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 includes processing the proliferated cell clumps to make them suitable for transplantation into the body.

[0209] 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 disease cells derived from a patient; b) perfusing 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 proliferating cell clump containing the target cells, wherein the proliferating cell clump contains capillary-like structures; and

[0210] d) The proliferated cell mass is transplanted into an experimental animal to obtain an experimental animal model for the disease.

[0211] In one aspect, the present invention provides the use of the in vitro proliferated cell clumps, the 3D cell cultures, or the 3D cell culture apparatus 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, organ engineering, etc. Example

[0212] Example 1: Construction and application of vascularized organoid 3D culture

[0213] 1.1. Isolation and Culture of Lung Cancer Organoids and Lung Cancer Associated Fibroblasts (LU-CAF): Lung cancer surgical samples were partially debrided (removing blood vessels, fat, etc.) using surgical scissors and forceps. The samples were digested with collagenase B (Roche, 11088807001), filtered through a 100 μm filter (Corning, 431752), and centrifuged at 1000 rpm, 4°C for 5 min. After centrifugation, the cells were resuspended in PBS and centrifuged at 1000 rpm, 4°C for 5 min. Finally, erythrocytes were lysed using erythrocyte lysis buffer (Thermo, A1049201) and centrifuged at 1000 rpm, 4°C for 5 min. A portion of the cell pellet was cultured in Matrigel (Corning, 356231) and lung cancer organoid culture medium (Xianjuebio). The remaining cell pellet was cultured in lung cancer associated fibroblast (LU-CAF) culture medium. Cells were cultured in the appropriate culture medium (Xianjuebio) for adherent growth of fibroblasts (LU-CAF). Once the cells reached a confluence of ≥90%, they were digested and used. Culture conditions were 37℃ and 5% CO2.

[0214] 1.2. Method for constructing 3D cultures of vascularized organoids:

[0215] (1) Take a 50mL centrifuge tube, add 30mL of sterile water, then add 12g of Pluronic F-127, place it in a 4℃ refrigerator for 24 hours and shake to dissolve, and let it stand for 3 hours to wait for the bubbles to disappear. Finally, prepare a solution with a mass concentration of 30-40% (w / v) and store it in the refrigerator for later use. Weigh 1g of GelMA (methacrylamide gelatin, 20% substitution, manufactured by Xianjue Biotechnology), place it in a 15mL centrifuge tube, add 10mL of deionized water, and then place it in a 50℃ water bath for 3-4 hours to dissolve and prepare a 10% GelMA solution. At the same time, weigh 150mg of LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, manufactured by Xianjue Biotechnology) using an electronic balance, place it in another 15mL centrifuge tube, add 3mL of deionized water, and shake with a vortex mixer for 10s. After the GelMA is completely dissolved, add 100μL of LAP solution, followed by 200μL of 0.1M NaOH, adjust the pH to 7, and finally filter the GM20 solution through a 0.22μm filter and place it in a 37℃ incubator for later use.

[0216] (2) Quickly take the Pluronic F-127 solution out of the refrigerator and slowly fill it into the printer material bucket. Then fill it into the printer, turn on the host, select to use the low temperature nozzle to print inside the homemade device, set the syringe temperature to 25°C, the base plate to 30°C, select to use a long needle with a diameter of 0.5mm, the air pressure parameter to 0.3MPa, the printing speed to 1mm / s, print at a line interval of 1.5mm, the printing angle to 60°, the printing layer height to 0.4mm, and print a total of 9 layers of structure. Print the skeleton structure in the central chamber of the microfluidic chip.

[0217] (3) Collect approximately 10,000 lung cancer organoids cultured above, add approximately 30,000 LU-CAF cells and 30,000 HUVEC cells (adding CAF cells or HUVEC cells alone to the culture system will not result in blood vessel formation), mix with Matrigel / Type I collagen at a 1:1 ratio, and then solidify onto an F127 scaffold. Finally, fill the device with the above-mentioned GelMa (XJ MATRIX, XJ-GM-20) solution until the entire device chamber is filled, and then cure by irradiation with a UV lamp for 3 minutes at an intensity of 10 W / cm². 2 The device was then sealed.

[0218] (4) Transfer the device to a 4°C refrigerator to cool for 20 minutes. After taking it out, use a syringe pump to connect the two sides of the chip to the inlet / outlet. Use culture medium at a rate of 20 mL / h to remove Pluronic F-127 by perfusing one side and extracting the other side.

[0219] (5) Take one HUVEC cell line, centrifuge it, and resuspend it in EGM-2 medium to a density of 10⁻⁶ cells / mL. 7 HUVEC cells were injected into the perfusion tube from one side of the chip using a syringe pump at a rate of 1 mL / min. The device was rotated 90° every 15 minutes for 4 hours until all HUVEC cells were attached to the tube wall. Subsequent steps were performed in a 37° incubator.

[0220] (6) Use an injection pump to connect the two sides of the chip to the inlet / outlet and continuously infuse the culture medium at a rate of 5 μL / min. The culture medium contains the following cytokines: EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Y-27632 and Wnt-3a to maintain cell and organoid viability and provide shear force to enable HUVEC cells to form a vascular barrier and maintain the recording cell state.

[0221] Example 2: Construction and application of vascularized spherical 3D culture bodies

[0222] 2.1 Spheroidization of Lung Cancer Cell Lines

[0223] A549-GFP (Shanghai Academy of Sciences) cells were cultured in F12K medium (Gibco, 31765035) containing 10% FBS (Gibco, 10099141c); HUVEC / HUVEC-RFP vascular endothelial cells (Zhongqiao Biotechnology) were cultured in ECM (Sciencell, 1001) medium. All culture conditions were 37℃ and 5% CO2. Mycoplasma detection was performed using a mycoplasma detection kit (Vazyme / D101-02). Cells were spheroidized in low-adsorption 96-well plates (Corning, 7007), with A549-GFP and HUVEC-RFP cell lines selected for spheroidization, and cultured overnight at 37℃.

[0224] 2.2 Method for constructing 3D cultures of vascularized lung cancer cell lines:

[0225] (1) Take a 50mL centrifuge tube, add 30mL of sterile water, then add 12g of Pluronic F-127, place it in a 4℃ refrigerator for 24 hours and shake to dissolve, and let it stand for 3 hours to wait for the bubbles to disappear. Finally, prepare a 40% (w / v) mass concentration solution and store it in the refrigerator for later use. Weigh 1g of GelMA (methacrylamide gelatin, manufactured by Xianjue Biotechnology), with a degree of substitution of 20%, and transfer it to a 15mL centrifuge tube. Add 10mL of deionized water and place the tube in a 50℃ water bath for 3-4 hours to dissolve and prepare a 10% GM20 solution. Simultaneously, weigh 150mg of LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, manufactured by Xianjue Biotechnology), transfer it to another 15mL centrifuge tube, add 3mL of deionized water, and shake the tube for 10 seconds. After the GelMA is completely dissolved, add 100μL of LAP solution, followed by 200μL of 0.1M NaOH to adjust the pH to 7. Finally, filter the GM20 solution through a 0.22μm filter and place it in a 37℃ incubator for later use.

[0226] (2) Quickly take the Pluronic F-127 solution out of the refrigerator and slowly fill it into the printer material bucket. Then fill it into the printer, turn on the host, select to use the low temperature nozzle to print inside the homemade device, set the syringe temperature to 25°C, the base plate to 30°C, select to use a long needle with a diameter of 0.5mm, the air pressure parameter to 0.3MPa, the printing speed to 1mm / s, print at a line interval of 1.5mm, the printing angle to 60°, the printing layer height to 0.4mm, and print a total of 9 layers of structure. Print the skeleton structure in the central chamber of the microfluidic chip.

[0227] (3) Collect overnight cultured lung cancer cell line A549-GFP / vascular endothelial cell HUVEC-RFP mixed spheroids, add LU-CAF, add about 30,000 LU-CAF and 30,000 HUVEC to 10,000 lung cancer cells, mix Matrigel / type I collagen 1:1, and then fix it on F127 scaffold.

[0228] (4) Transfer the device to a 4°C refrigerator to cool for 20 minutes. After taking it out, use a syringe pump to connect the two sides of the chip to the inlet / outlet. Use culture medium at a rate of 20 mL / h to remove Pluronic F-127 by perfusing one side and extracting the other side.

[0229] (5) Take one HUVEC cell line, centrifuge it, and resuspend it in EGM-2 medium to a density of 10⁻⁶ cells / mL. 7HUVEC cells were injected into the perfusion tube from one side of the chip using a syringe pump at a rate of 1 mL / min. The device was rotated 90° every 15 minutes for 4 hours until all HUVEC cells were attached to the tube wall. This step was carried out in a 37°C incubator.

[0230] (6) Use an injection pump to connect the two sides of the chip to the inlet / outlet and continuously infuse the culture medium at a rate of 5 μL / min. The culture medium contains the following cytokines: EGF, FGF7 / 10, VEGFA, Noggin, RSPO1, Y-27632 and Wnt-3a to maintain cell and organoid viability and provide shear force to enable HUVEC cells to form a vascular barrier and maintain the recording cell state.

[0231] The main manufacturers and models of the materials and instruments used in the above embodiments are as follows:

[0232] The bio-3D printer was purchased from Hangzhou Genofi Company.

[0233] Pluronic F127 purchased from SigmaAldrich

[0234] Type I collagen is Type I collagen (rat tail type I collagen) was purchased from Corning Incorporated, product number: 354236.

[0235] GelMA (methacrylamide gelatin) is a double-bond modified gelatin that can form a hydrogel through UV and visible light crosslinking under the presence of a photoinitiator. GelMA hydrogels possess excellent biocompatibility and tunable mechanical properties, making them widely applicable in biomedical fields such as tissue engineering, 3D printing, and drug delivery. This product was purchased from Suzhou Xianjue New Materials Technology Co., Ltd.

[0236] All culture media were purchased from Suzhou Xianjue Biotechnology Co., Ltd.

[0237] Example 3: Cultivation and Evaluation of 3D Cultures

[0238] 3.1 The state of the above-mentioned vascularized lung cancer cell line 3D culture and organoid 3D culture was observed under a microscope.

[0239] The state of the 3D culture of lung cancer cell lines and organoids was observed under a microscope on the third day of culture. The results are shown in Figure 2. In Figure 2, 2A is the lung cancer cell line labeled with green fluorescent protein (A549-GFP), B is the vascular endothelial cells labeled with red fluorescent protein (HUVEC-RFP), which can confirm the formation of capillary-like structural network between tumor cell spheres. C is the cell state under bright field.

[0240] The state of lung cancer organoids was observed on the third day of culture, and the results are shown in Figure 3. In Figure 3, A is the organoid labeled with green fluorescent protein, B is the blood vessel labeled with red fluorescent protein, and C is the superimposed effect. Arrow 1 points to the blood vessel and arrow 2 points to the organoid. It can be observed that there is obvious angiogenesis in the organoid.

[0241] Figure 4 shows the state of the 3D organoid culture on the third day of culture. The arrow points to the area where translucent 3D spherical organoids are generated.

[0242] 3.2 Western blot analysis of protein immunoblotting to detect the expression of lung cancer, extracellular matrix, epithelial-mesenchymal transition, and vascular marker proteins

[0243] After culturing the 3D culture bodies for three days, proteins were collected and subjected to Western blotting experiments. It was found that compared with the 3D cell model of single-cell culture (control group: only target cells were cultured without the addition of HUVEC, CAF and other cells for co-culture, and other conditions were the same), the vascularization model was able to retain the expression of lung cancer markers and promote the expression of extracellular matrix markers, epithelial-mesenchymal transition markers and vascular markers.

[0244] Figure 5 shows the results of detecting lung cancer marker proteins (CK7, TTF-1), extracellular matrix marker proteins (MMP2, COL1A1, FN, Vimentin), and vascularization-related marker protein (CD31) on day 3 of culturing 3D lung cancer cell lines and 3D lung cancer organoids, respectively. It can be seen that the preparation method of this invention, compared with the control group, promotes ECM protein expression, while tumor proteins remain intact, and it also promotes the expression of proteins that promote microvascular formation. These characteristics are closer to those of natural tumor tissue.

[0245] 3.3 Hematoxylin / eosin staining (HE staining) for observing the morphology and structure of vascularized cell lines / organoids

[0246] HE staining was performed on sections of 3D culture of lung cancer cell line. The results are shown in Figure 6. Arrow 1 points to the nucleus of lung cancer cell line, arrow 2 points to the cytoplasm, and arrow 3 points to the extracellular matrix. The tissue structure of fusion between blood vessels and between blood vessels and lung cancer cell tissue can be clearly seen.

[0247] Figure 8 shows the HE staining results and immunohistochemical staining results for CD31, FN, CK7, and Vimentin in 3D cultures of lung cancer organoids. In the HE row, arrow 1 points to the nucleus, arrow 2 to the cytoplasm, and arrow 3 to the extracellular matrix. In the CD31 row, the arrows indicate that CD31 was lowly expressed in the cytoplasm and cell membrane on days 1 and 3 of culture. In the CK7 row, the arrows indicate that CK7 was highly expressed in the cytoplasm and cell membrane on days 1 and 3 of culture. In the FN row, the arrows indicate that FN was lowly expressed in the cell membrane on days 1 and 3 of culture. In the Vimentin row, the arrows indicate that Vimentin was highly expressed in the cytoplasm and cell membrane on days 1 and 3 of culture.

[0248] The magnified HE staining image is shown in Figure 9. Arrow 1 points to blood vessels (including tiny capillaries), arrow 2 points to vascular endothelial tissue, and arrow 3 points to tumor tissue or tumor nests. This tissue section confirms primary organoid tumor tissue of lung cancer, with fused vascular structures. Scattered cell clumps exist against an eosinophilic background, exhibiting structures similar to those of natural tumors. The fusion of large and small blood vessels / capillaries is clearly visible, and the CD31 immunohistochemical staining in Figure 8 indicates the fusion of tumor tissue and blood vessels, with positive staining of vascular endothelial cells surrounding the cavitary structures.

[0249] 3.4 Immunohistochemical observation of lung cancer, extracellular matrix, epithelial-mesenchymal transition, and expression of vascular marker proteins

[0250] Figure 7 shows the immunohistochemical results of CD31, CK7, FN, and Vimentin in the 3D culture of lung cancer cell line. It can be seen that in the 3D culture of lung cancer cell line, FN is expressed at low levels on the cell membrane, CD31 is expressed at high levels on the cell membrane, and CK7 and Vimentin are expressed at high levels on the cytoplasm and cell membrane.

[0251] Figure 8 shows the immunohistochemical results of CD31, CK7, FN, and Vimentin in 3D lung cancer organoid cultures. It can be seen that CD31 is highly expressed on the cell membrane, FN is lowly expressed on the cell membrane, and CK7 and Vimentin are highly expressed on the cytoplasm and cell membrane. A mixed system of tumor and capillary formation is observed, with a layer of CD31-positive vascular endothelial cells surrounding the lumen (vascular-like structures). FN, CK7, and Vimentin represent extracellular matrix markers and tumor markers, respectively. This demonstrates that the tumor culture model of this invention can promote ECM protein expression while maintaining tumor proteins and promoting the expression of proteins that promote microvascular formation. Such a tumor culture model can not only be cultured for a long time but also possesses biological characteristics similar to the natural state, providing an immune microenvironment further similar to the natural state.

[0252] Example 4: Specific Embodiment of the Chip Device

[0253] Figure 10 illustrates one implementation of the microfluidic chip, and Figure 11 shows a schematic diagram of the fixture, where 1 represents the fixture cover, 2 represents the chip cover, 3 represents the chip body, and 4 represents the fixture base. The following is an example of preparing and culturing 3D cultures using the microfluidic chip shown in Figure 11:

[0254] (1) Take a 50mL centrifuge tube, add 30mL of sterile water, then add 12g of Pluronic F-127, place it in a 4℃ refrigerator for 24 hours and shake to dissolve, and let it stand for 3 hours to wait for the bubbles to disappear. Finally, prepare a 40% (w / v) mass concentration solution and store it in the refrigerator for later use. Weigh 1g of GelMA (methacrylamide gelatin, 20% substitution) from Xianjue Biotechnology, transfer it to a 15mL centrifuge tube, add 10mL of deionized water, and place it in a 50℃ water bath for 3-4 hours to dissolve and prepare a 10% GM20 solution. Simultaneously, weigh 150mg of LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, manufactured by Xianjue Biotechnology) using an electronic balance, transfer it to another 15mL centrifuge tube, add 3mL of deionized water, and shake for 10 seconds. After the GelMA is completely dissolved, add 100μL of LAP solution, followed by 200μL of 0.1M NaOH, adjust the pH to 7, and finally filter the GM20 solution through a 0.22μm filter and place it in a 37℃ incubator.

[0255] (2) Quickly take the Pluronic F-127 solution out of the refrigerator and slowly fill it into the printer material bucket. Then fill it into the printer, turn on the host, select to use the low temperature nozzle to print inside the homemade device, set the syringe temperature to 25°C, the base plate to 30°C, select to use a long needle with a diameter of 0.5mm, the air pressure parameter to 0.3MPa, the printing speed to 1mm / s, print at a line interval of 1.5mm, the printing angle to 60°, the printing layer height to 0.4mm, and print a total of 9 layers of structure. Print the skeleton structure in the central chamber of the microfluidic chip.

[0256] (3) Collect overnight cultured lung cancer cell line A549-GFP / vascular endothelial cell HUVEC-RFP mixed spheroids, add LU-CAF, and fix them onto F127 scaffolds with type I collagen (Corning, 354236). Finally, fill the device with the above-mentioned Gelma (XJ MATRIX, XJ-GM-20) solution until the entire device chamber is filled, and then cure with UV lamp for 3 min at an intensity of 10 W / cm². 2 The device was then sealed.

[0257] (4) Transfer the device to a 4°C refrigerator to cool for 20 minutes. After taking it out, use a syringe pump to connect the two sides of the chip to the inlet / outlet. Use culture medium at a rate of 20 mL / h to remove Pluronic F-127 by perfusing one side and extracting the other side.

[0258] (5) Take one HUVEC cell line, centrifuge it, and resuspend it in EGM-2 medium to a density of 10⁻⁶ cells / mL. 7 HUVEC cells were injected into the perfusion tube from one side of the chip using a syringe pump at a rate of 1 mL / min. The device was rotated 90° every 15 minutes for 4 hours until all HUVEC cells were attached to the tube wall. This step was carried out in a 37°C incubator.

[0259] (6) Use an injection pump to connect the two sides of the chip to the inlet / outlet and continuously infuse the culture medium at a rate of 5 μL / min to maintain cell and organoid viability, while providing shear force to enable HUVEC cells to form a vascular barrier and maintain the cell state.

[0260] Example 5: Application of chip device in anti-angiogenic drug testing

[0261] [Detailed Rules 91, 27.06.2025] According to the methods provided in Examples 1-4, a chip device was prepared using lung cancer organoids, lung cancer CAF, and HUVEC cells as materials, and used for anti-angiogenic drug testing. Lenvatinib, an anti-angiogenic drug, was applied. Five days after application, the length of blood vessels was analyzed using ImageJ software to evaluate the inhibitory effect of lenvatinib on blood vessels. The results are shown in Figure 16, where A is the negative control group, B is the positive control group, C is the drug application group with a concentration of 300 ng / mL, D is the drug application group with a concentration of 33 ng / mL, and E is the drug application group with a concentration of 3.7 ng / mL. The inhibition rate results of different drug concentration groups are shown in F. The inhibitory effect on blood vessels was lowest at a drug concentration of 33 ng / mL, and the inhibition rate was highest at a drug concentration of 300 ng / mL, reaching 59.50%, showing a relatively significant inhibitory effect on blood vessels. Therefore, the chip device provided by this invention can be used to test candidate drugs.

[0262] Equivalent form and scope

[0263] Those skilled in the art will recognize or be able to determine many equivalent forms of the specific embodiments of the invention described herein using only conventional experiments. The scope of the invention is not intended to be limited by the foregoing description, but rather as shown in the following claims.

[0264] The use of sequential terms (e.g., "first", "second", "third", etc.) that modify claim elements in the claims does not imply any priority, superiority, or that one claim element is in order over another or in the order of the method action. Rather, they serve only as labels to distinguish one claim element with a certain name from another element with the same name (but using sequential terms), thereby differentiating the claim elements.

[0265] Unless explicitly stated otherwise, the articles “a” and “an” used in the specification and claims as used herein shall be understood to include plural indicators. Unless explicitly stated otherwise in the text, a claim or description including “or” among one or more members of the group shall be considered to satisfy the presence, adoption, or relevance of one, more, or all members of the group. This invention includes embodiments in which only one member of the group is present, adopted, or relevant to a given product or method. This invention also includes embodiments in which more than one or all members of the group are present, adopted, or relevant to a given product or method. Furthermore, it is understood that, unless otherwise stated or unless it would clearly cause contradiction or inconsistency to a person skilled in the art, this invention covers all variations, combinations, and permutations of one or more limiting elements, phrases, descriptive modifiers, etc., of one or more illustrated claims introduced into another claim (or any other related claim) dependent on the same basic claim. When elements are represented as a list (e.g., in a Markush group or similar format), it is understood that it also discloses the subgroups of the elements, and any element may be removed from the group. Generally, it should be understood that when references are made to the invention or its various aspects to include specific elements, features, etc., certain embodiments of the invention or its various aspects are composed of or substantially composed of these elements, features, etc. For the sake of simplicity, those embodiments will not be described in every instance with the same level of detail described herein. It should be understood that any embodiment or aspect of the invention may be expressly excluded by the claims, whether or not such a specific exclusion is stated in the description. Publications, websites, or other references describing the background of the invention and providing further detailed information about its implementation are incorporated herein by reference.

Claims

1. A method for preparing a 3D cell culture, comprising the steps of: (A) constructing a scaffold based on a sacrificial material; (B) arranging first culture cells and a solidifiable biomedium around the scaffold, and then solidifying the biomedium to form a solidified biomedium block; (C) covering the biomedium block with a solidifiable encapsulation material, and then solidifying the encapsulation material so that the biomedium block is encapsulated and reinforced; and (D) removing the scaffold, thereby forming a perfusion structure inside the biomedium block to obtain the 3D cell culture. The sacrificial material comprises one or more selected from the group consisting of a heat-sensitive material, a light-sensitive material, or a combination thereof.

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

3. The production method of a 3D cell culture body according to claim 1 or 2, wherein, At least a portion of the scaffold is constructed by 3D printing.

4. The method for producing a 3D cell culture body according to any one of claims 1 to 3, wherein The first culture cells comprise target cells; and optionally one or more cells selected from the group consisting of stromal cells and vascular-like cells.

5. The method for producing a 3D cell culture body according to any one of claims 1 to 4, wherein 6. The method for preparing a 3D cell culture according to any one of claims 1-5, wherein the target cells comprise one or more selected from the group consisting of cardiac tissue cells, brain tissue cells, intestinal tissue cells, stomach tissue cells, pancreatic tissue cells, muscle tissue cells, lung tissue cells, liver tissue cells, kidney tissue cells, spleen tissue cells, or tumor tissue cells. 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.

7. The method of producing a 3D cell culture body according to any one of claims 1 to 6, wherein The stromal cells are selected from fibroblast stromal cells or mesenchymal stem cells; and the vascular-like cells are selected from vascular endothelial cells or vascular-like organs.

8. The method of producing a 3D cell culture body according to any one of claims 1 to 7, wherein, 9. The method for preparing a 3D cell culture according to any one of claims 1-8, wherein The stromal cells account for 10-90%, preferably 20-70%, more preferably 30-50% of the first culture cells; and / or The ratio of stromal cells to target cells in the first culture cells is 1:3-10:1; preferably 1:3-5:1; more preferably 1:2-3:1, more preferably 3:1; and / or The vascular-like cells account for 10-90%, preferably 20-70%, more preferably 30-50% of the first culture cells; and / or The ratio of vascular-like cells to target cells in the first culture 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 1-9, 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. The biomedium comprises one or more selected from the group consisting of a heat-sensitive material, a light-sensitive material, or a combination thereof.

11. The method of producing a 3D cell culture body according to any one of claims 1 to 10, wherein, ​ 12. The method of producing a 3D cell culture body according to any one of claims 1 to 11, wherein, The biological medium comprises a protein-based gel, a synthetic-based gel, a polysaccharide-based gel, and / or a matrix-based gel.

13. The method for preparing a 3D cell culture body according to claim 12, wherein, The protein-based gel comprises one or more selected from the group consisting of collagen I, fibrin, gelatin, elastin, fibroin, or a combination thereof.

14. The production method of a 3D cell culture body according to claim 12, wherein, The synthetic-based gel comprises one or more selected from the group consisting of polyethylene glycol, polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), poly(N-isopropyl acrylamide), poly(ethylene oxide), polyglycolic acid, or a combination thereof.

15. The preparation method of the 3D cell culture body as claimed in claim 12, wherein, The polysaccharide-based gel comprises one or more selected from the group consisting of hyaluronic acid, alginate, chitosan, dextran, chondroitin sulfate.

16. The preparation method of the 3D cell culture body as claimed in claim 12, wherein, The matrix-based 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 of producing a 3D cell cultivator according to any one of claims 1 to 16, wherein, The biological medium further comprises a cell-inducing agent.

18. The method of producing a 3D cell cultivator of any one of claims 17, wherein, The cell-inducing agent comprises a cytokine.

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

20. The method of producing a 3D cell cultivator according to any one of claims 1 to 19, wherein, Before the step (D), the distance between the center lines of two adjacent segments of the scaffold in the block of the biological medium is no more than 2000 microns.

21. The method of producing a 3D cell cultivator according to any one of claims 1 to 20, wherein, The encapsulating material comprises one or more selected from the group consisting of a heat-sensitive material, a light-sensitive material, or a combination thereof.

22. The method of producing a 3D cell cultivator of any one of claims 2-21, wherein, The light-sensitive material is a photocured gel.

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

24. The method of preparing a 3D cell culture according to any one of claims 1-23, wherein the encapsulating material has a higher strength after solidification than the biological medium after solidification; optionally, the encapsulating material has any one or more of the following characteristics: a storage modulus of 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 80000-120000 Pa, more preferably 90000-110000 Pa; and / or a loss modulus of 2000-50000 Pa, preferably 5000-45000 Pa, more preferably 10000-40000 Pa, more preferably 20000-30000 Pa; optionally, the encapsulating material has a higher strength after solidification than the biological medium after solidification means that the storage modulus and / or the loss modulus of the biological medium is lower than that of the encapsulating material; and optionally, the biological medium and / or the encapsulating material is biodegradable. The biological medium is collagen I or Matrigel, and the encapsulating material is GelMA.

25. The method of producing a 3D cell cultivator according to any one of claims 1 to 24, wherein, The step (D) comprises removing the scaffold at a temperature ranging from 0-37 °C, optionally, the specific temperature is 0-10 °C, preferably 3-6 °C, more preferably 4 °C.

26. The method of producing a 3D cell cultivator of any one of claims 1-25, wherein, ​ 27. The method of producing a 3D cell cultivator of any one of claims 1-26, wherein, The perfusion structure has a three-dimensional network of pipe cavities.

28. The method of producing a 3D cell cultivator of any one of claims 1-27, wherein, The maximum distance between adjacent pipes in the three-dimensional network of pipe cavities is no more than 400 microns.

29. The method of producing a 3D cell cultivator of any one of claims 1-28, wherein, The longest distance from any position of the biomedia block to the perfusion structure is no more than 200 microns.

30. The method of producing a 3D cell cultivator of any one of claims 1-29, wherein, The diameter of the pipes formed by the perfusion structure is 100-2000 microns.

31. The method of any one of claims 1-30, further comprising the step of: (E) seeding a second culture cell within the perfusion structure, allowing the second culture cell to attach to the inner wall of the perfusion structure.

32. The production method of a 3D cell culture body according to claim 31, wherein The second culture cell comprises vascular endothelial cells, tumor-associated fibroblasts.

33. The method of any one of claims 1-32, further comprising the step of: (F) continuously perfusing a culture medium into the perfusion structure, allowing the target cells to proliferate in a three-dimensional manner in the 3D cell culture, resulting in a proliferated cell mass containing target cells.

34. The method of claim 33, wherein the proliferated cell mass contains a newly formed vascular network structure, the vascular network structure having one or more of the following characteristics: having large vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters no more than 10 microns; having vessels expressing CD31 protein; having a plurality of vessels interconnected to form a network; the vascular network structure being fused with the target cells.

35. [Rule 91, 08.04.2025] The method of any one of claims 33-34, wherein the proliferated cell mass expresses at least one target tissue molecular marker that is specifically expressed in a target tissue from which the target cells are derived.

36. A 3D cell culture prepared by the method of any one of claims 1-35.

37. An in vitro cultured 3D cell culture comprising: a solidified biomedia block having a three-dimensional network of perfusion structures inside the biomedia block; and an encapsulation material covering the outer layer of the solidified biomedia block; wherein having a vascular barrier comprising vascular endothelial cells on the inner wall of the perfusion structure and a cell mass comprising target cells in the biomedia block adjacent to the perfusion structure, the cell mass containing a newly formed vascularized network, the vascularized network having one or more of the following characteristics: having large vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters no more than 10 microns; having vessels expressing CD31; having a plurality of vessels interconnected to form a network; the vascular network structure being fused with the target cells.

38. The 3D cell culture of claim 36 or 37, wherein the cell mass expresses at least one target tissue molecular marker that is specifically expressed in a 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 tissue of interest molecular marker comprises a lung cancer marker, a lung cancer extracellular matrix marker, or a blood vessel marker.

41. The 3D cell culture of claim 40, wherein: the lung cancer marker comprises one or more markers selected from the group consisting of CK-7 and TTF-1; the lung cancer extracellular matrix marker comprises one or more markers selected from the group consisting of FN, Vimentin, COL1A1, MMP2; and / or the blood vessel marker comprises CD31. the cell mass expresses at least Vimentin, FN, and CK-7 simultaneously. the marker is detected by immunohistochemistry or immunofluorescence. the lung cancer extracellular matrix marker is detected by immunohistochemistry at the cell membrane.

42. The 3D cell cultivator of claim 41, wherein, the solidified biomedia mass comprises a solidified protein-based gel, a solidified synthetic-based gel, a solidified polysaccharide-based gel, and / or a solidified matrix-based gel.

43. The 3D cell cultivator of any one of claims 38-42, wherein, the solidified biomedia mass further comprises stromal cells and / or a cell inductive agent.

44. The 3D cell cultivator of claim 43, wherein, the cell inductive agent comprises a cytokine.

45. The 3D cell cultivator of any one of claims 36-44, wherein, the encapsulation material is as defined in any one of claims 20-24.

46. The 3D cell cultivator of any one of claims 36-45, wherein, the perfusion structure is formed by a sacrificial material-based method.

47. The 3D cell cultivator of claim 46, wherein, the perfusion structure can allow perfusion of culture medium to allow the cell mass to proliferate in three dimensions in the 3D cell culture.

48. The 3D cell cultivator of any one of claims 36-47, wherein, the 3D cell culture can allow at least one cell mass to be cultured in at least 1 cubic centimeter.

49. The 3D cell cultivator of any one of claims 36-48, wherein, the 3D cell culture can allow at least one cell mass to be cultured for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks.

50. The 3D cell cultivator of any one of claims 36-49, wherein, the solidified biomedia mass contains at least one cell mass having a size of at least 10 cubic millimeters and at least one of the following properties:

51. The 3D cell cultivator of any one of claims 36-50, wherein, expresses the tissue of interest molecular marker; 52. The 3D cell cultivator of any one of claims 36-51, wherein, has at least one region containing a newly formed vascularized network having one or more of the following characteristics:

53. The 3D cell cultivator of any one of claims 36-52, wherein, has large blood vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters not exceeding 10 microns; has blood vessels expressing CD31; has a plurality of blood vessels interconnected to form a network; and / or the vascular network structure is integrated with the target cells.

54. The 3D cell culture of any one of claims 36-53, wherein the cell mass has a size of at least 5 cubic millimeters, at least 50 cubic millimeters, at least 100 cubic millimeters, at least 200 cubic millimeters, at least 300 cubic millimeters, at least 500 cubic millimeters, at least 700 cubic millimeters, at least 800 cubic millimeters, at least 900 cubic millimeters, or at least 1 cubic centimeter.

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

56. A cell mass proliferated in vitro, which is obtained by culturing the 3D cell culture of any one of claims 36-55. ​ ​ ​ 57. A 3D cell culture device having the 3D cell culture body of any one of claims 36-55 therein.

58. The 3D cell-culture device of claim 57, wherein, The 3D cell culture device further has a housing that encloses the 3D cell culture body.

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

60. The 3D cell-culture device of claim 59, wherein, The device capable of applying mechanical force is capable of rotating the 3D cell culture body.

61. The 3D cell-culture device of any one of claims 57-60, wherein, The inlet and / or outlet of the perfusion 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 of any one of claims 61 or 62, wherein, The inlet and / or outlet of the perfusion structure is further provided with a device for removing air bubbles between the pump.

64. The 3D cell-culture device of any one of claims 61-63, wherein, The pump and / or the device capable of applying mechanical force is controlled by a control system.

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

66. A method of testing a candidate drug, comprising: (a) providing the 3D cell culture body of any one of claims 36-55 or the 3D cell culture device of any one of claims 57-65; (b) perfusing the candidate drug into the perfusion structure of the 3D cell culture body, (c) determining the biological activity of the cell aggregate in the 3D cell culture body after perfusion of the candidate drug to determine a change compared to the biological activity before perfusion, and (d) assessing 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 connections, and / or cell morphology changes of the cell aggregate.

69. A method of culturing a tissue, comprising: (1) providing the 3D cell culture body of any one of claims 36-55 or the 3D cell culture device of any one of claims 57-65; (2) perfusing a culture medium into the perfusion structure of the 3D cell culture body to allow the cell aggregate to proliferate in three dimensions; and (3) obtaining a proliferated cell aggregate, wherein the proliferated cell aggregate contains a newly formed vascularized network having one or more of the following characteristics: having large blood vessels with diameters ranging from 10 microns to 100 microns and capillaries with diameters not exceeding 10 microns; having blood vessels expressing CD31; having a plurality of blood vessels interconnected to form a network; the vascular network structure is fused with target cells.

70. The method of claim 69, further comprising processing the proliferated cell aggregate so that it is suitable for implantation into a body.

Images