Method for constructing vascularized organoid spheroid model, vascularized organoid spheroid model and use thereof

Abstract

The present invention provides a method for constructing a vascularized organoid spheroid model, a vascularized organoid spheroid model, and a use thereof. The construction method comprises the following steps: S100: preparing or acquiring an organoid, subjecting the organoid to digestion to obtain organoid cells, and acquiring fibroblasts and vascular endothelial cells; S200: mixing the organoid cells, the fibroblasts and the vascular endothelial cells, and resuspending with a culture medium, to obtain an organoid multicellular suspension; S300: inoculating the organoid multicellular suspension into a culture chamber having a U-shaped bottom, or a culture well having a U-shaped bottom, performing spheroid culture, and self-assembling, to form a vascularized organoid spheroid model. The present disclosure uses co-culture of organoid cells, fibroblasts and vascular endothelial cells, and also uses a low-adhesion U-shaped well plate to form microspheres, so as to prepare the vascularized organoid spheroid aggregate model.

Description

Method for constructing a vascularized organoid spheroid model, vascularized organoid spheroid model and its application Technical Field

[0001] The present disclosure relates to the field of bioengineering technology, and in particular to a method for constructing a vascularized organoid spheroid model, a vascularized organoid spheroid model, and applications thereof. Background Art

[0002] Cancer is a highly structured, complex ecosystem that includes tumor cells and a large number of non-tumor cells, all embedded in an extracellular matrix. The tumor microenvironment (TME) includes a variety of immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, and a variety of other tissue-resident cells. This complex environment directly or indirectly affects the phenotype of tumor cells through complex reciprocal signaling mechanisms mediated by soluble factors, intercellular contacts, and extracellular matrix (ECM) remodeling, leading to high tumor heterogeneity. TME cells are involved in every stage of cancer progression, affecting tumor occurrence, progression, invasion, intravasation, metastatic spread, and growth. TME cells and the molecules they secrete are believed to play a key role in the pathogenesis of cancer and represent attractive therapeutic targets. The complex interactions between tumor cells and mediators are crucial for the rational development of effective anticancer drugs.

[0003] Cancer heterogeneity significantly contributes to diverse patient responses to treatment, including therapeutic resistance, and this complexity has become a considerable obstacle to uncovering the specific mechanisms of treatment failure. Organotypic models of patient-specific tumors have the potential to transform our understanding of cancer heterogeneity and its impact on personalized medicine. These advances are due in part to the ability of organoid models to stably preserve the genetic, proteomic, morphological, and druggable characteristics of the parent tumor in vitro, while also providing unprecedented genomic and environmental manipulation. However, it is difficult for tumor organoid models to fully characterize the original tissue because they typically lack a complete microenvironment. Tumor organoids and the tumor microenvironment can be likened to "seeds and soil." Constructing tumor organoid models with a microenvironment for drug testing will be closer to the human body's true response to drug action.

[0004] Angiogenic inhibitors are an important class of anti-tumor drugs. In 1971, Professor Folkman proposed the theory of tumor angiogenesis, pointing out that both local tumor growth and distant metastasis are inseparable from the generation of tumor angiogenesis. The growth and metastasis of solid tumors undergo a transformation process from the pre-vascular phase to the vascular phase. Tumor angiogenic inhibitors can inhibit the formation of the tumor angiogenic growth factor VEGF, thereby inducing the natural apoptosis of vascular endothelial cells and destroying the tumor's new vascular network. However, traditional in vitro models make it difficult to detect the anti-tumor effects of such drugs. Tumor models such as tumor cell models, tumor organoid models, and tumor aggregate models contain only tumor cells and do not respond to angiogenic inhibitors. Vascular models can only reflect the effects on angiogenesis and cannot further reflect the effects on tumors.

[0005] Summary of the Invention

[0006] In view of this, the present disclosure provides a method for constructing a vascularized organoid spheroid model, a vascularized organoid spheroid model and its application. The vascularized organoid spheroid model constructed by the construction method described in the present disclosure has a dense structure and strong simulation, which is closer to the real reflection of the human body's response to drug effects. It can be used for the screening of anti-tumor drugs and the sensitivity testing of anti-tumor drugs, and can also be used for toxicity testing of non-tumor drugs.

[0007] In a first aspect, the present disclosure provides a method for constructing a vascularized organoid spheroid model, the construction method comprising the following steps: S100: preparing or obtaining an organoid, wherein the organoid is a primary organoid or a passaged organoid, digesting the primary organoid or the passaged organoid to obtain organoid cells, and obtaining fibroblasts and vascular endothelial cells. S200: mixing the organoid cells, the fibroblasts, and the vascular endothelial cells, and resuspending them in a culture medium to obtain an organoid multicellular suspension. S300: inoculating the organoid multicellular suspension into a culture chamber or a culture well, performing spheroid culture, and self-assembling to form the vascularized organoid spheroid model.

[0008] The organoid is an organoid constructed from tissue extraction or an organoid differentiated from iPS stem cells, and the organoid can be a primary organoid or an organoid to be passaged.

[0009] For example, in some embodiments, the organoid multicellular suspension is inoculated into a culture chamber having a U-shaped bottom for sphere formation culture; in other embodiments, the organoid multicellular suspension is inoculated into a culture well having a U-shaped bottom for sphere formation culture. Preferably, the culture chamber and / or the culture well are low-adhesion U-shaped culture chambers and / or low-adhesion U-shaped culture wells to facilitate subsequent self-assembly into spheres.

[0010] The method for constructing the vascularized organoid spheroid model disclosed in the present invention first mixes the organoid cells obtained by digestion of primary organoids or passaged organoids with fibroblasts and vascular endothelial cells in an appropriate proportion, and then sequentially performs multiple stages of spheroid culture. During the culture process, a culture vessel with a U-shaped bottom is used to facilitate the formation of a tightly structured spheroid structure. The organoid cells used in the present invention can, on the one hand, truly reflect the physiological characteristics of tumor tissue and non-tumor tissue. On the other hand, the organoid cells can proliferate and passage, so that the simulation of the prepared vascularized organoid spheroid model is better. Moreover, the present invention uses an appropriate amount of fibroblasts and an appropriate amount of vascular endothelial cells to co-culture with the organoid cells, and can grow an organoid microenvironment composed of fibroblast structure and microvessels, further improving the simulation of the prepared vascularized organoid spheroid model, so that the prepared vascularized organoid spheroid model can better characterize the original tissue. The construction method disclosed herein uses spheroid culture plus cell culture scaffold materials (such as hydrogels, matrix gels, and other cell culture gels) to facilitate the observation of cell migration and the study of tumor invasion in subsequent drug sensitivity testing.

[0011] In some embodiments, the total number of the organoid cells, the fibroblasts, and the vascular endothelial cells is 5000 to 2*10 4 The diameter of the constructed vascularized organoid spheroid model is in the range of 250 μm to 800 μm. The total number of cells is within the above range and matches the appropriate model diameter, which easily forms a necrotic core inside the model, which is conducive to the gradient increase of the proliferation degree of the model cells from the inside to the outside and the gradual increase of the oxygen content of the model from the inside to the outside, and the simulation is better. Preferably, the diameter of the constructed vascularized organoid spheroid model is in the range of 300 μm to 600 μm. More preferably, the diameter of the constructed vascularized organoid spheroid model is in the range of 350 μm to 450 μm.

[0012] In some embodiments, the organoid is a primary tumor organoid or a passaged tumor organoid, which is digested into single cells to obtain tumor organoid cells, the fibroblasts include tumor-associated fibroblasts, and the cell number ratio of the tumor organoid cells, the tumor-associated fibroblasts, and the vascular endothelial cells is 1:(0.1-1):(0.05-1). When the cell number ratio of the three cells is within the above range, the resulting vascularized organoid spheroid model has a denser structure and closer connections between cells, which is conducive to improved simulation.

[0013] Unless otherwise specified in the present disclosure, the culture medium includes at least components for culturing organoid cells and components for culturing vascular endothelial cells.

[0014] In some embodiments, the culture medium is a mixed culture medium comprising at least a first culture medium for culturing the organoid cells and a second culture medium for culturing the vascular endothelial cells. In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2). In this case, it is more conducive to the co-culture of organoid cells, fibroblasts, and vascular endothelial cells, and is conducive to the growth and reproduction of vascularized organoid spheroids. Preferably, the mass ratio of the first culture medium to the second culture medium is 1:(0.8-1.2).

[0015] In some embodiments, the sphere formation culture includes a first stage sphere formation culture and a second stage sphere formation culture performed sequentially. The first stage sphere formation culture includes: inoculating the organoid multicellular suspension into the culture chamber with a U-shaped bottom or the culture well with a U-shaped bottom for agglomeration treatment, so that the cells in the organoid multicellular suspension are agglomerated, and then placed in a culture environment for culture for 1 to 5 days to obtain an organoid sphere aggregate model intermediate with clear boundaries and microvessels, and then performing the second stage sphere formation culture. Among them, the culture time of the first stage is preferably 2 to 3 days. It should be noted that in this disclosure, unless otherwise specified, the culture environment refers to a standard culture environment of 37°C and 5% CO2. Preferably, the agglomeration treatment includes centrifugation at 950rpm to 1050rpm for 5min to 6min.

[0016] In some embodiments, the conditions for the second stage of spheroid culture include: discarding the original culture medium in the culture chamber or culture well containing the organoid spheroid model intermediate, adding a cell culture scaffold material, adding the mixed culture medium after gelation, and continuing to culture in a culture environment for 1 to 2 days. Preferably, the second stage of culture time is 1 day.

[0017] In other embodiments, the conditions for the second stage spheroidization culture include: discarding the original culture medium in the culture chamber or the culture well, adding a cell culture scaffold material, transferring the cells to the drug sensitivity chip culture chamber or the drug sensitivity chip culture well, wherein the drug sensitivity chip culture chamber and the drug sensitivity chip culture well have a U-shaped bottom and are interconnected, adding the mixed culture medium after gelation, placing the cells in a culture environment for dynamic culture, and the second stage culture time is 1 to 2 days. Preferably, the second stage culture time is 1 day.

[0018] In some embodiments, the dynamic culture conditions include: performing flow perfusion culture in a culture environment at a rocking speed of 3° to 10° for 15 to 120 minutes. For example, in some embodiments, perfusion culture is performed in a culture environment at a rocking angle of 3° / 120 minutes; in other embodiments, perfusion culture is performed in a culture environment at a rocking angle of 7° / 30 minutes; and in other embodiments, perfusion culture is performed in a culture environment at a rocking angle of 7° / 15 minutes.

[0019] In some embodiments, the organoid is selected from any one of primary lung cancer organoids, iPS stem cell-differentiated lung cancer organoids, or passaged lung cancer organoids, and the primary lung cancer organoids, iPS stem cell-differentiated lung cancer organoids, or passaged lung cancer organoids are digested into single cells to obtain lung cancer organoid cells, the fibroblasts include lung cancer-associated fibroblasts, and the cell number ratio of the lung cancer organoid cells, the lung cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.4-0.8):(0.1-0.5). In this case, the mixed culture medium includes at least a first culture medium for culturing the lung cancer organoid cells and a second culture medium for culturing the vascular endothelial cells, and the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

[0020] Exemplarily, the lung cancer organoid cells, the lung cancer-associated fibroblasts, and the vascular endothelial cells are mixed at a cell number ratio of 1: (0.4-0.8): (0.1-0.5), and resuspended in the above-mentioned mixed culture medium to obtain a lung cancer organoid multicellular suspension, which is inoculated into a culture chamber or culture well with a U-shaped bottom for the above-mentioned two-stage sphere culture. Alternatively, after the first stage of sphere culture is completed, the lung cancer organoid multicellular suspension is transferred to a drug sensitivity chip culture chamber or drug sensitivity chip culture well, and the above-mentioned mixed culture medium is added after gelation, and placed in a culture environment for the second stage of sphere culture.

[0021] In some embodiments, the organoid is a primary colorectal cancer organoid or a passaged colorectal cancer organoid, and the primary colorectal cancer organoid or the passaged colorectal cancer organoid is digested into a single cell to obtain colorectal cancer organoid cells, the fibroblasts include colorectal cancer-associated fibroblasts, and the cell number ratio of the colorectal cancer organoid cells, the colorectal cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.1-0.8):(0.2-0.6). At this time, the mixed culture medium includes at least a first culture medium for culturing the colorectal cancer organoid cells and a second culture medium for culturing the vascular endothelial cells, and the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

[0022] Exemplarily, the colorectal cancer organoid cells, the colorectal cancer-associated fibroblasts, and the vascular endothelial cells are mixed and resuspended in the above-mentioned mixed culture medium to obtain a colorectal cancer organoid multicellular suspension. The colorectal cancer organoid multicellular suspension is then inoculated into a culture chamber or culture well with a U-shaped bottom for the above-mentioned two-stage sphere formation culture. Alternatively, after the first stage of sphere formation culture is completed, the colorectal cancer organoid multicellular suspension is transferred to a drug sensitivity chip culture chamber or culture well, and after gelation, the above-mentioned mixed culture medium is added, and the suspension is placed in a culture environment for the second stage of sphere formation culture.

[0023] In some embodiments, the organoid is a primary pancreatic cancer organoid or a passaged pancreatic cancer organoid, and the primary pancreatic cancer organoid or the passaged pancreatic cancer organoid is digested into single cells to obtain pancreatic cancer organoid cells, the fibroblasts include pancreatic cancer-associated fibroblasts, and the cell number ratio of the pancreatic cancer organoid cells, the pancreatic cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.2-0.7):(0.2-0.7). In this case, the mixed culture medium includes at least a first culture medium for culturing the pancreatic cancer organoid cells and a second culture medium for culturing the vascular endothelial cells, and the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

[0024] Exemplarily, the pancreatic cancer organoid cells, the pancreatic cancer-associated fibroblasts, and the vascular endothelial cells are mixed and resuspended in the above-mentioned mixed culture medium to obtain a pancreatic cancer organoid multicellular suspension. The pancreatic cancer organoid multicellular suspension is then inoculated into a culture chamber or culture well with a U-shaped bottom for the above-mentioned two-stage sphere formation culture. Alternatively, after the first stage of sphere formation culture is completed, the pancreatic cancer organoid multicellular suspension is transferred to a drug sensitivity chip culture chamber or culture well, and after gelation, the above-mentioned mixed culture medium is added, and the pancreatic cancer organoid multicellular suspension is placed in a culture environment for the second stage of sphere formation culture.

[0025] In some embodiments, the organoid is a primary non-tumor organoid or a passaged non-tumor organoid, and the primary non-tumor organoid or the passaged non-tumor organoid is digested into a single cell to obtain a non-tumor organoid cell, and the non-tumor organoid is selected from any one of a liver organoid, a lung organoid, a colorectal organoid, or a pancreatic organoid, and the fibroblasts include related fibroblasts, for example, liver-related fibroblasts, lung-related fibroblasts, colorectal-related fibroblasts, or pancreatic-related fibroblasts. The non-tumor organoid cells, the related fibroblasts, and the vascular endothelial cells are mixed according to a cell number ratio of 1: (1 to 2): (1 to 2), and resuspended in a mixed culture medium to obtain a non-tumor organoid multicellular suspension. At this time, the mixed culture medium includes at least a first culture medium for culturing the non-tumor organoid cells and a second culture medium for culturing the vascular endothelial cells, and the mass ratio of the first culture medium to the second culture medium is 1: (0.5 to 2).

[0026] In some embodiments, the non-tumor organoid multicellular suspension is inoculated into a culture chamber or culture well with a U-shaped bottom for the two stages of sphere formation culture described above. In other embodiments, after the first stage of sphere formation culture is completed, the non-tumor organoid multicellular suspension is transferred to a drug sensitivity chip culture chamber or culture well, and after gelation, mixed culture medium is added, and the suspension is placed in a culture environment for the second stage of sphere formation culture.

[0027] In the construction method disclosed in the present invention, the organoid multicellular suspension is first inoculated into a U-shaped culture cavity or a U-shaped culture well, and then the first stage sphere formation culture and the second stage sphere formation culture are carried out in sequence. After the first stage sphere formation culture, an organoid spheroid aggregate model intermediate with clear boundaries and microvessels can be obtained, and then the cell culture scaffold material is continued to be added to the U-shaped culture cavity or the U-shaped culture well containing the organoid spheroid aggregate model intermediate. After gelation, the second stage sphere formation culture is carried out. The second stage sphere formation culture can be a static culture directly placed in the culture environment, or a dynamic culture placed in the culture environment. It can be selected according to actual conditions, and the present invention does not limit it. After the second stage sphere formation culture of the present invention with the addition of a matrix, the microvessels continue to grow to form a large-area complex network, which is beneficial for observing cell migration during subsequent drug testing, and the simulation is better. In some embodiments, the U-shaped culture cavity is a low-adhesion U-shaped culture cavity; the U-shaped culture well is a low-adhesion U-shaped culture well.

[0028] In a second aspect, the present disclosure provides a vascularized organoid spheroid model constructed using the aforementioned construction method. The vascularized organoid spheroid model has a spheroid structure within which multiple organoids aggregate and grow, and wherein microvessels radiate from the inside out. The vascularized organoid spheroid model also includes an organoid microenvironment composed of fibroblasts and microvessels.

[0029] In some embodiments, the vascularized organoid spheroid model has cell proliferation, and the degree of cell proliferation increases gradually from the inside to the outside, and the oxygen content of the vascularized organoid spheroid model gradually increases from the inside to the outside.

[0030] In some embodiments, the diameter of the vascularized organoid spheroid model ranges from 250 μm to 800 μm. Preferably, the diameter of the vascularized organoid spheroid model ranges from 300 μm to 600 μm.

[0031] In a third aspect, the present disclosure provides an application of the above-mentioned vascularized organoid spheroid model in detection, wherein the detection includes any one of drug screening, drug sensitivity detection, drug toxicity detection or non-drug substance toxicity detection.

[0032] In some embodiments, the application includes at least the following steps: adding a test drug to a culture chamber or culture well containing the vascularized organoid spheroid model, culturing the model in a culture environment for 3 to 5 days, and detecting changes in indicators of the vascularized organoid spheroid model. The indicator changes include at least one of changes in shape, size, activity, vascular morphology, and protein expression or distribution in the vascularized organoid spheroid model.

[0033] In other embodiments, the application includes at least the following steps: adding a test drug to a culture chamber or well of a drug sensitivity chip containing the vascularized organoid spheroid model, dynamically culturing the model in a culture environment for 3 to 5 days, and detecting changes in indicators of the vascularized organoid spheroid model. The indicator changes include at least one of changes in shape, size, activity, vascular morphology, and protein expression or distribution of the vascularized organoid spheroid model.

[0034] In a fourth aspect, the present disclosure provides a drug detection method, which includes the following steps: S100: preparing or obtaining organoids, digesting the organoids to obtain organoid cells, obtaining fibroblasts and vascular endothelial cells, S200: mixing the organoid cells, fibroblasts and vascular endothelial cells, and resuspending them in a culture medium to obtain an organoid multicellular suspension, S300: inoculating the organoid multicellular suspension into a culture chamber or culture well for spheroid culture, and self-assembling to form the vascularized organoid spheroid model, S400: adding the drug to be tested to the culture chamber or culture well containing the vascularized organoid spheroid model, placing it in a culture environment for culture, and detecting changes in indicators of the vascularized organoid spheroid model. The selection of organoids and specific parameters can refer to the above three aspects and will not be repeated here.

[0035] In a fifth aspect, the present disclosure provides a culture medium composition comprising a first culture medium for culturing organoid cells and a second culture medium for culturing vascular endothelial cells, wherein the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

[0036] In some preferred embodiments, the mass ratio of the first culture medium to the second culture medium is 1:(0.8-1.2).

[0037] The organoid cells can refer to the contents of the first to third aspects and will not be described in detail.

[0038] Compared with the prior art, the technical solution disclosed herein has at least the following technical effects: The present disclosure constructs a vascularized organoid spheroid model by co-culturing organoid cells, fibroblasts, and vascular endothelial cells. During the culture process, multiple stages of spheroid culture are performed in a U-shaped bottom culture chamber or well to form a dense spheroid structure. Furthermore, by regulating the cell number ratio of the three cell types within an appropriate range, the present disclosure achieves closer intercellular connections in the prepared vascularized organoid spheroid model, better simulation, and the ability to fully characterize the original tissue. Compared with the prior art models, the present disclosure is more suitable for detection, particularly for the detection of anti-tumor angiogenesis-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0040] FIG1 is a test diagram of the vascularized lung cancer organoid spheroid model prepared in Example 1 of the present disclosure;

[0041] FIG2 is a comparative test diagram comparing the digestion of lung cancer organoids into single cells and the non-digestion of lung cancer organoids into single cells during the preparation of a vascularized lung cancer organoid spheroid model in Example 1 of the present disclosure;

[0042] FIG3 is a comparative test diagram of changing the cell number ratio between cells in the process of preparing a vascularized lung cancer organoid spheroid model in Example 1 of the present disclosure;

[0043] FIG4 is an observation diagram of the vascularized lung cancer organoid spheroid model prepared in Example 1 of the present disclosure during drug sensitivity testing;

[0044] FIG5 is a graph showing the results of detecting the lysates of the three groups of experiments in FIG4 using a chemiluminescence microplate reader;

[0045] FIG6 is an observation diagram of the vascularized lung cancer organoid spheroid model prepared in Example 2 of the present disclosure during drug sensitivity testing;

[0046] FIG7 is a graph showing the results of detecting the lysate in FIG6 using a chemiluminescence microplate reader;

[0047] FIG8 is an observation diagram of the vascularized colorectal cancer organoid spheroid model prepared in Example 3 of the present disclosure during drug sensitivity testing;

[0048] FIG9 is a graph showing the results of detecting the lysate in FIG8 using a chemiluminescence microplate reader;

[0049] FIG10 is an observation diagram of the vascularized pancreatic cancer organoid spheroid model prepared in Example 4 of the present disclosure during drug sensitivity testing;

[0050] FIG11 is a graph showing the results of detecting the lysate in FIG10 using a chemiluminescence microplate reader;

[0051] FIG12 is an observation diagram of the vascularized liver organoid spheroid model prepared in Example 5 of the present disclosure during the hepatotoxicity test;

[0052] FIG13 is a graph showing the results of detecting the lysates of the three groups of experiments in FIG12 using a chemiluminescence microplate reader;

[0053] FIG14 is a schematic diagram of the structure of the drug sensitivity chip used in the present disclosure. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present disclosure and are not intended to limit the present disclosure.

[0055] Method for constructing a vascularized lung cancer organoid spheroid model

[0056] S100: preparing or obtaining lung cancer organoids, digesting the lung cancer organoids to obtain lung cancer organoid cells, and obtaining fibroblasts and vascular endothelial cells;

[0057] The lung cancer organoid cells are selected from tissue-extracted lung cancer organoids and iPS stem cell-differentiated lung cancer organoids, and can be either primary lung cancer organoids or passaged lung cancer organoids.

[0058] Preferably, the lung cancer organoid cells are selected from lung cancer organoid cells obtained by digesting primary lung cancer organoids into single cells or lung cancer organoid cells obtained by digesting passaged lung cancer organoids into single cells.

[0059] S200: mixing the lung cancer organoid cells, the fibroblasts, and the vascular endothelial cells, and resuspending them in a culture medium to obtain a lung cancer organoid multicellular suspension;

[0060] The cell number ratio of the lung cancer organoid cells, the fibroblasts, and the vascular endothelial cells is 1:(0.1-1):(0.05-1);

[0061] Preferably, the fibroblasts include the lung cancer-associated fibroblasts, and the cell number ratio of the lung cancer organoid cells, the lung cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.4-0.8):(0.1-0.5);

[0062] The culture medium is a mixed culture medium comprising at least a first culture medium for culturing the lung cancer organoids and a second culture medium for culturing the vascular endothelial cells. The mass ratio of the first culture medium to the second culture medium is 1:(0.5-2). Preferably, the mass ratio of the first culture medium to the second culture medium is 1:(0.8-1.2).

[0063] S300: inoculating the multicellular suspension of the lung cancer organoid into a culture chamber or a culture well for spheroid culture to form a vascularized lung cancer organoid spheroid model through self-assembly.

[0064] Specifically, the multicellular suspension of the lung cancer organoid is inoculated into a culture chamber with a U-shaped bottom or a culture well with a U-shaped bottom for sphere culture, and the sphere culture includes sequentially performing the first stage sphere culture and the second stage sphere culture. The conditions for the first stage sphere culture include: inoculating the multicellular suspension of the lung cancer organoid into a culture chamber with a U-shaped bottom or a culture well with a U-shaped bottom, centrifuging at 950rpm to 1050rpm for 5min to 6min, and placing it in a culture environment at 37°C and 5% CO2 for culture. The culture time of the first stage sphere culture is 1 to 5 days. The above centrifugation step is only an example, and other methods can also be used to make the cells cluster, such as group modification on the chip. After the first stage of sphere culture, a lung cancer organoid sphere aggregate model intermediate with clear boundaries and microvessels is obtained, and then the second stage of sphere culture is carried out.

[0065] In some embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix gel, adding the mixed culture medium after gelation, and placing the culture medium in a culture environment at 37°C and 5% CO2 for 1 to 2 days.

[0066] In other embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix glue, transferring to the drug sensitivity chip culture chamber or the drug sensitivity chip culture well, adding the mixed culture medium after gelation, placing it in a culture environment of 37°C and 5% CO2 for dynamic culture, and the culture time is 1 to 2 days. The drug sensitivity chip culture chamber and the sensitive chip culture well both have a U-shaped bottom and are interconnected.

[0067] The dynamic culture conditions include: performing flow perfusion culture in a culture environment at a rocking speed of 3° to 10° for 15 to 120 minutes. For example, flow perfusion culture is performed in a culture environment at 37°C and 5% CO2 at a rocking angle of 3° / 120 minutes; or, performing flow perfusion culture in a culture environment at 37°C and 5% CO2 at a rocking angle of 7° / 30 minutes; or, performing flow perfusion culture in a culture environment at 37°C and 5% CO2 at a rocking angle of 7° / 15 minutes. Regarding the dynamic culture conditions, the following examples are the same and will not be repeated.

[0068] Method for constructing a vascularized colorectal cancer organoid spheroid model

[0069] S100: Prepare or obtain organoids, wherein the organoids are primary colorectal cancer organoids or passaged colorectal cancer organoids, digest the primary colorectal cancer organoids or the passaged colorectal cancer organoids into single cells to obtain colorectal cancer organoid cells, and obtain colorectal cancer-associated fibroblasts and vascular endothelial cells.

[0070] S200: mixing the colorectal cancer organoid cells, the colorectal cancer-associated fibroblasts, and the vascular endothelial cells, and resuspending them in a culture medium to obtain a colorectal cancer organoid multicellular suspension;

[0071] The cell number ratio of the colorectal cancer organoid cells, the colorectal cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.1-0.8):(0.2-0.6);

[0072] The culture medium is a mixed culture medium, which includes at least a first culture medium for culturing the colorectal cancer organoid cells and a second culture medium for culturing the vascular endothelial cells. In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:(0.5~2).

[0073] S300: inoculating the colorectal cancer organoid multicellular suspension into a culture chamber or culture well for spheroid culture to self-assemble into a vascularized colorectal cancer organoid spheroid model.

[0074] Specifically, the colorectal cancer organoid multicellular suspension is inoculated into a culture chamber with a U-shaped bottom or a culture well with a U-shaped bottom for sphere culture, and the sphere culture includes a first stage sphere culture and a second stage sphere culture performed sequentially. In some embodiments, the first stage sphere culture includes: inoculating the colorectal cancer organoid multicellular suspension into the culture chamber or the culture well with a U-shaped bottom, centrifuging at 800-1200 rpm for 5-10 minutes, and placing it in a culture environment at 37°C and 5% CO2 for culture. The culture time of the first stage sphere culture is 1 to 5 days. After the first stage of sphere culture, a colorectal cancer organoid sphere aggregate model intermediate with clear boundaries and microvessels is obtained, and then the second stage sphere culture is performed.

[0075] In some embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix gel, adding the mixed culture medium after gelation, and continuing to culture in a culture environment at 37°C and 5% CO2 for 1 to 2 days.

[0076] In other embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix glue, transferring to the drug sensitivity chip culture chamber or the drug sensitivity chip culture well, adding the culture medium after gelation, placing it in a culture environment for dynamic culture, and the culture time is 1 to 2 days. The drug sensitivity chip culture chamber or the sensitive chip culture well has a U-shaped bottom and is interconnected.

[0077] Method for constructing a vascularized pancreatic cancer organoid spheroid model

[0078] S100: preparing or obtaining organoids, wherein the organoids are primary pancreatic cancer organoids or passaged pancreatic cancer organoids, digesting the primary pancreatic cancer organoids or the passaged pancreatic cancer organoids into single cells to obtain pancreatic cancer organoid cells, and obtaining pancreatic cancer-associated fibroblasts and vascular endothelial cells.

[0079] S200: mixing the pancreatic cancer organoid cells, the pancreatic cancer-associated fibroblasts, and the vascular endothelial cells, and resuspending them in a culture medium to obtain a pancreatic cancer organoid multicellular suspension;

[0080] The cell number ratio of the pancreatic cancer organoid cells, the pancreatic cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.2-0.7):(0.2-0.7);

[0081] The culture medium is a mixed culture medium, which includes at least a first culture medium for culturing the pancreatic cancer organoid cells and a second culture medium for culturing the vascular endothelial cells. In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:(0.5~2).

[0082] S300: inoculating the pancreatic cancer organoid multicellular suspension into a culture chamber or culture well for spheroid culture to self-assemble into a vascularized pancreatic cancer organoid spheroid model.

[0083] Specifically, the pancreatic cancer organoid multicellular suspension is inoculated into a culture chamber with a U-shaped bottom or a culture well with a U-shaped bottom for sphere culture, and the sphere culture includes a first stage sphere culture and a second stage sphere culture performed sequentially. In some embodiments, the first stage sphere culture includes: inoculating the pancreatic cancer organoid multicellular suspension into the culture chamber or the culture well with a U-shaped bottom, centrifuging at 800-1200 rpm for 5-10 minutes, and placing it in a culture environment at 37°C and 5% CO2 for culture. The culture time of the first stage sphere culture is 1 to 5 days, and a pancreatic cancer organoid sphere aggregate model intermediate with clear boundaries and microvessels is obtained, and then the second stage sphere culture is performed.

[0084] In some embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix gel, adding the mixed culture medium after gelation, and placing the culture medium in a culture environment at 37°C and 5% CO2 for 1 to 2 days.

[0085] In other embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix glue, transferring to the drug sensitivity chip culture chamber or the drug sensitivity chip culture well, adding the mixed culture medium after gelation, placing it in a culture environment of 37°C and 5% CO2 for dynamic culture, and the culture time is 1 to 2 days. The drug sensitivity chip culture chamber or the sensitive chip culture well has a U-shaped bottom and is interconnected.

[0086] Method for constructing a vascularized liver organoid spheroid model

[0087] S100: preparing or obtaining organoids, wherein the organoids are primary liver organoids or passaged liver organoids, digesting the primary liver organoids or passaged liver organoids into single cells to obtain liver organoid cells, and obtaining liver-associated fibroblasts and vascular endothelial cells.

[0088] S200: mixing the liver organoid cells, the liver-associated fibroblasts, and the vascular endothelial cells, and resuspending them in a culture medium to obtain a liver organoid multicellular suspension;

[0089] The cell number ratio of the liver organoid cells, the liver-associated fibroblasts, and the vascular endothelial cells is 1:(1-2):(1-2);

[0090] The culture medium is a mixed culture medium, which includes at least a first culture medium for culturing the liver organoid cells and a second culture medium for culturing the vascular endothelial cells. The mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

[0091] S300: inoculating the multicellular suspension of the liver organoid into a culture chamber or culture well for spheroid culture to form a vascularized liver organoid spheroid model through self-assembly.

[0092] Specifically, the multicellular suspension of the liver organoid is inoculated into a culture chamber with a U-shaped bottom or a culture well with a U-shaped bottom for sphere culture, and the sphere culture includes a first stage sphere culture and a second stage sphere culture performed sequentially. In some embodiments, the first stage sphere culture includes: inoculating the multicellular suspension of the liver organoid into the culture chamber or the culture well with a U-shaped bottom, centrifuging at 800-1200 rpm for 5-10 minutes, placing it in a culture environment for culture for 1-5 days, obtaining a liver organoid spheroid model intermediate with clear boundaries and microvessels, and then performing the second stage sphere culture.

[0093] In some embodiments, the conditions for the second stage of sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding a cell culture scaffold material such as hydrogel or matrix gel, adding the culture medium after gelation, and placing the culture medium in the culture environment for continued culture, and the culture time is 1 to 2 days.

[0094] In other embodiments, the conditions for the second stage sphere culture include: discarding the original culture medium in the culture chamber or the culture well, adding cell culture scaffold materials such as hydrogel and matrix glue, transferring to the drug sensitivity chip culture chamber or the drug sensitivity chip culture well, adding the culture medium after gelation, placing it in a culture environment for dynamic culture, and the culture time is 1 to 2 days. The drug sensitivity chip culture chamber or the sensitive chip culture well has a U-shaped bottom and is interconnected.

[0095] Example 1

[0096] Preparation of a vascularized lung cancer organoid spheroid model (forming a vascularized lung cancer organoid spheroid model on a U-shaped well plate)

[0097] S100: Obtain lung cancer organoid cells, fibroblasts, and vascular endothelial cells.

[0098] Step 1. Select primary or passaged lung cancer organoids and add pre-chilled PBS to each well of a culture plate. Collect the pellet into a centrifuge tube and refrigerate at 4°C to 6°C for 15 to 20 minutes. Centrifuge and remove the supernatant to obtain a pellet of lung cancer organoids.

[0099] Step 2: Add Tryple E and digest for 5-10 minutes. After digestion into single cells, centrifuge and remove the supernatant. Add PBS and centrifuge again to remove excess enzyme solution to obtain a precipitate. Add mixed culture medium and resuspend. Count the cells to determine the cell concentration of the lung cancer organoid suspension.

[0100] Wherein, in the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

[0101] Step 3: Take lung cancer-associated fibroblasts, digest them into single cells, resuspend them in the above-mentioned mixed culture medium, and count them;

[0102] Step 4: Take the vascular endothelial cells, digest them into single cells, resuspend them in the above mixed culture medium, and count them.

[0103] S200: The lung cancer organoid cells, fibroblasts, and vascular endothelial cells are mixed and resuspended in a culture medium to obtain a lung cancer organoid multicellular suspension.

[0104] Step 5: Mix the lung cancer organoid cells, lung cancer-associated fibroblasts, and vascular endothelial cells at a cell number ratio of 1:(0.4-0.8):(0.1-0.5), resuspend them in the above-mentioned mixed culture medium, and obtain a lung cancer organoid multicellular suspension.

[0105] S300: Inoculate the multicellular suspension of lung cancer organoids into a culture chamber or culture well for spheroid culture, and self-assemble to form a vascularized lung cancer organoid spheroid model.

[0106] Step 6: Add the multicellular suspension of lung cancer organoids to a low-adhesion U-bottom culture well and centrifuge at 950-1050 rpm for 5-6 minutes.

[0107] Step 7: Place the cells in a 37°C, 5% CO2 culture environment for the first stage of sphere formation;

[0108] Step 8: After 1 to 5 days of culture, the original culture medium in the well plate is removed, and cell culture scaffold materials such as hydrogel and matrix gel are added. After gelation, the above-mentioned mixed culture medium is added and placed in a culture environment at 37°C and 5% CO2 for the second stage of sphere formation culture. The second stage of sphere formation culture lasts for 1 to 2 days.

[0109] Example 1 Drug Testing:

[0110] (1) Prepare drug-containing culture medium of appropriate concentration;

[0111] (2) Transferring the vascularized lung cancer organoid spheroid model formed by the above culture to a 96-well plate;

[0112] (3) Observe and record the growth status of the vascularized lung cancer organoid spheroid model, remove the original culture medium in the 96-well plate, add 0.01-100 μM drug-containing culture medium to each well, and culture in a 37°C, 5% CO2 incubator;

[0113] (4) Observe and record the growth status of the vascularized lung cancer organoid spheroid model on day 3;

[0114] (5) On day 5, the growth status of the vascularized lung cancer organoid spheroid model was observed and recorded, and activity was detected using a CTG detection kit;

[0115] Add 80-120 μL of CTG detection reagent to each well, shake on a shaker for 5-10 minutes, and incubate at room temperature for 20-30 minutes;

[0116] (6) After incubation, the cells were detected using a chemiluminescence microplate reader.

[0117] Example 2

[0118] Preparation of a vascularized lung cancer organoid spheroid model (forming a vascularized lung cancer organoid spheroid model on a drug sensitivity chip)

[0119] S100: Obtain lung cancer organoid cells, fibroblasts, and vascular endothelial cells.

[0120] Step 1. Select primary or passaged lung cancer organoids and add 1 mL to 2 mL of pre-chilled PBS to each well of a culture plate. Collect the pellet into a centrifuge tube and refrigerate at 4°C to 6°C for 15 to 20 minutes. Centrifuge and remove the supernatant to obtain a pellet of lung cancer organoids.

[0121] Step 2: Add Tryple E and digest for 5-10 minutes. After digestion into single cells, centrifuge and remove the supernatant. Add PBS and centrifuge again to remove excess enzyme solution to obtain a precipitate. Add mixed culture medium and resuspend. Count the cells to determine the cell concentration of the lung cancer organoid suspension.

[0122] Among them, the mixed culture medium in Example 2 is the same as the mixed culture medium in Example 1 and is not described again.

[0123] Step 3: Take lung cancer-associated fibroblasts, digest them into single cells, resuspend them in the above-mentioned mixed culture medium, and count them;

[0124] Step 4: Take the vascular endothelial cells, digest them into single cells, resuspend them in the above mixed culture medium, and count them.

[0125] S200: The lung cancer organoid cells, fibroblasts, and vascular endothelial cells are mixed and resuspended in culture medium to obtain a lung cancer organoid multicellular suspension.

[0126] Step 5: Mix the lung cancer organoid cells, the lung cancer-associated fibroblasts, and the vascular endothelial cells at a cell number ratio of 1:(0.4-0.8):(0.1-0.5), and resuspend them in the above-mentioned mixed culture medium to obtain a lung cancer organoid multicellular suspension.

[0127] S300: Inoculate the multicellular suspension of lung cancer organoids into a culture chamber or culture well for spheroid culture, and self-assemble to form a vascularized lung cancer organoid spheroid model.

[0128] Step 6: Add the multicellular suspension of lung cancer organoids to a low-adhesion U-bottom culture chamber and centrifuge at 950-1050 rpm for 5-6 minutes.

[0129] Step 7: Place the cells in a culture environment at 37°C and 5% CO2 for the first stage of sphere formation culture;

[0130] Step 8: After 1 to 5 days of culture, the original culture medium in the well plate is removed, and a cell culture scaffold material such as a hydrogel or matrigel is added. The spheroids are transferred to the middle well of the drug sensitivity chip. After gelation, 25 μL to 35 μL of mixed culture medium is added to the middle well of the drug sensitivity chip, and 45 μL to 55 μL of mixed culture medium is added to the wells on both sides of the drug sensitivity chip. The drug sensitivity chip is placed in a rocking perfusion instrument and cultured by flow perfusion at a speed of 3° to 10° for 15 to 120 minutes in a culture environment at 37°C and 5% CO2. The culture time is 1 to 2 days.

[0131] Example 2 drug test:

[0132] (1) Prepare drug-containing culture medium of appropriate concentration;

[0133] (2) Observe and record the growth status of the vascularized lung cancer organoid spheroid model in the culture chamber of the drug sensitivity chip, and remove the original culture medium in the culture chamber;

[0134] (3) Add 45-55 μL of drug-containing culture medium to the liquid storage chambers on both sides of the drug sensitivity chip (corresponding to the holes on both sides of the drug sensitivity chip), and add 25-35 μL of drug-containing culture medium to the middle culture chamber (corresponding to the middle hole of the drug sensitivity chip);

[0135] (4) Place the drug-sensitive chip after drug addition into a rocking perfusion instrument and set the conditions to 3° / 120 min. Then place the rocking perfusion instrument into an incubator at 37°C and 5% CO2 for flow perfusion culture.

[0136] (5) On the third day, the growth status of the vascularized lung cancer organoid spheroid model was observed and recorded, and 25–35 μL of drug-containing culture medium was continuously added to the middle culture chamber;

[0137] (6) On day 5, the growth status of the vascularized lung cancer organoid spheroid model was observed and recorded, and activity was detected using a CTG detection kit;

[0138] (7) Discard all drug-containing culture medium in the reservoirs on both sides and add 20-30 μL CTG diluent (1:1 dilution of CTG reagent stock solution and mixed culture medium);

[0139] (8) Without discarding the drug-containing culture medium in the middle culture chamber, directly add 45-55 μL of CTG stock solution and mix thoroughly by pipetting;

[0140] (9) Shake on a shaker for 5–10 min and incubate at room temperature for 25–30 min.

[0141] (10) All lysates were taken out and placed in a 96-well plate with completely opaque sides and bottom, and detected using a chemiluminescence enzyme reader.

[0142] The incubation time during drug testing can be set to shorter or longer times depending on the drug's action and metabolism rate.

[0143] It should be noted that the construction of other models disclosed herein, such as vascularized colorectal cancer organoid spheroid models, vascularized pancreatic cancer organoid spheroid models, and other non-tumor organoid models, can refer to the aforementioned exemplary methods. The difference is that different types of organoid cells and fibroblasts are selected during the mixing process of organoid cells, fibroblasts, and vascular endothelial cells, and the corresponding cell number ratios are also different. For other purposes, the aforementioned exemplary construction methods can be referred to. Furthermore, drug testing for other models can refer to the testing procedures of Example 1 or Example 2, differing only in the type and concentration of drug in the drug-containing culture medium.

[0144] Hereinafter, examples and comparative examples are given to more specifically illustrate the embodiments of the present disclosure. The reagents and equipment used in the following examples and comparative examples can be purchased through commercial channels.

[0145] Alternatively, the lung cancer organoid culture kit was purchased from Jiangsu Aiweide Biotechnology Co., Ltd. with the catalog number KLU0101;

[0146] The colorectal cancer organoid culture kit was purchased from Bozhen Biotechnology (Suzhou) Co., Ltd. with the item number K2103-CR, the pancreatic cancer organoid culture kit was purchased from Jiangsu Avid Biotechnology Co., Ltd. with the item number KPA0101, and the liver organoid culture kit was purchased from Jiangsu Avid Biotechnology Co., Ltd. with the item number KLV0101.

[0147] Non-small cell lung cancer organoid culture medium (i.e., the first culture medium) was purchased from Jiangsu Aiweide Biotechnology Co., Ltd. with the catalog number KCJ-6-1;

[0148] Human primary lung cancer-associated fibroblasts were purchased from Hunan Meisen Biotechnology Co., Ltd. with the catalog number CTCC-258-Hum;

[0149] Human umbilical vein endothelial cells (HUVECs) were purchased from the Union Cell Resource Center with the catalog number 1101HUM-PUMC000437;

[0150] Colorectal cancer organoid culture medium was purchased from Jiangsu Avid Biotechnology Co., Ltd. with the catalog number KCJ-2, and pancreatic cancer organoid culture medium was purchased from Jiangsu Avid Biotechnology Co., Ltd. with the catalog number KCJ-3.

[0151] The low-adsorption U-bottom 96-well plate was purchased from Shanghai Noning Biotechnology Co., Ltd. with the catalog number NBH1296.

[0152] Example 1

[0153] (I) Preparation of a vascularized lung cancer organoid spheroid model (forming a vascularized lung cancer organoid spheroid model on a U-shaped well plate)

[0154] Step 1. Select primary lung cancer organoids (i.e., the lung cancer organoid culture kit with catalog number KLU0101, the same as in Example 2), add pre-chilled PBS to the culture well plate, add 1 mL of PBS to each well, collect the cells into a centrifuge tube, refrigerate at 4°C for 15 minutes, centrifuge (1000 rpm, 5 minutes), and remove the supernatant to obtain a lung cancer organoid pellet;

[0155] Step 2: Add Tryple E for 8 minutes to digest into single cells, then centrifuge (1000 rpm, 5 minutes). Remove the supernatant, add PBS, and centrifuge again (1000 rpm, 5 minutes) to remove excess enzyme solution. The precipitate is resuspended in mixed culture medium and the cells are counted to determine the cell concentration of the lung cancer organoid suspension.

[0156] Wherein, in the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:0.8, which is the same as that in Example 2 and will not be repeated herein.

[0157] Step 3: Primary human lung cancer-associated fibroblasts (i.e., lung cancer-associated fibroblasts, the same below) were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0158] Step 4: Human umbilical vein endothelial cells (HUVECs) were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0159] Step 5: Lung cancer organoid cells, primary human lung cancer-associated fibroblasts, and human umbilical vein endothelial cells (HUVECs) were mixed at a cell number ratio of 1:0.75:0.25 (the total number of cells per well was 5000, the number of lung cancer organoid cells was 2500 / well, the number of human primary lung cancer-associated fibroblasts was 1875 / well, and the number of human umbilical vein endothelial cells was 625 / well) to obtain a lung cancer organoid multicellular suspension;

[0160] Step 6: Add the above-mentioned lung cancer organoid multicellular suspension to a low-adhesion U-bottom 96-well plate and centrifuge at 1000 rpm for 5 minutes;

[0161] Step 7: Place the cells in an incubator at 37°C and 5% CO2 for the first stage of sphere formation culture;

[0162] Step 8. After 3 days of culture, remove the original culture medium in the well plate, add 5uL of hydrogel, and after gelation, add 100uL of the above mixed culture medium and place it in an incubator at 37°C and 5% CO2 for the second stage of sphere formation culture. The second stage of sphere formation culture lasts for 2 days.

[0163] The diameter of the vascularized lung cancer organoid spheroid model prepared in Example 1 is 400 μm.

[0164] FIG1 is a test of the vascularized lung cancer organoid spheroid model prepared in Example 1 of the present disclosure.

[0165] Figure 1a is a bright field image of the vascularized lung cancer organoid spheroid model. It can be seen that the vascularized lung cancer organoid spheroid model prepared in Example 1 has clear boundaries and grows large areas of microvessels with a complex network structure. Figure 1b uses green cell tracking dye (cell tracker) to mark lung cancer organoids. From the lung cancer organoid distribution map, it can be seen that lung cancer organoids grow uniformly in a spherical shape. Figure 1c is an unstained paraffin section of lung cancer organoids. It can be seen that the spheroid cells grow densely and the peripheral endothelial cells grow in a tubular shape. Figure 1d is a hematoxylin and eosin staining of paraffin sections of lung cancer organoids. It can be seen that lung cancer organoid cells fuse and grow with lung cancer-associated fibroblasts and vascular endothelial cells. The three cells promote each other to form spheroids. Figure 1e is a fluorescent image of microvessels generated in multicellular spheroids of lung cancer organoids. Green cell tracking dye is used to mark lung cancer organoids. The multicellular spheroids of lung cancer organoids are frozen and sectioned. Green fluorescence shows that organoids are distributed in spheroids, and that organoids fuse and grow with endothelial cells and fibroblasts. It can be seen from the test diagram of Figure 1 that the vascularized lung cancer organoid spheroid model prepared in Example 1 of the present disclosure has good simulation performance.

[0166] Figure 2 shows a comparison of the two situations of digesting lung cancer organoids into single cells and not digesting lung cancer organoids into single cells in the process of preparing a vascularized lung cancer organoid spheroid model in Example 1 of the present disclosure. Figure 2(a) shows the state of the lung cancer organoids after being digested into single cells and then mixed with other cells in the process of obtaining lung cancer organoid cells, and cultured on the 1st and 5th days. Figure 2(b) shows the state of the lung cancer organoids after being directly mixed with other cells in the process of obtaining lung cancer organoid cells, and cultured on the 1st and 5th days. It can be seen from the comparison photos in Figure 2 that it is easier to form spheroids after digesting lung cancer organoids into single cells and then mixing with other cells to form multicellular spheroids. At the same time, after 5 days of growth of the spheroids, it can be seen that the microvessels of the model are more abundant when the organoids are digested into single cells and then formed into multicellular spheroids.

[0167] FIG3 further compares the cell number ratios of lung cancer organoid cells, lung cancer-associated fibroblasts, and vascular endothelial cells in the process of preparing the vascularized lung cancer organoid spheroid model in Example 1 of the present disclosure. In FIG3(a), the cell number ratio of lung cancer organoid cells, lung cancer-associated fibroblasts, and vascular endothelial cells is 4:3:1, and the bright field image is obtained after mixing and culturing for 3 days. In FIG3(b), the cell number ratio of lung cancer organoid cells, lung cancer-associated fibroblasts, and vascular endothelial cells is 30:1:2, and the bright field image is obtained after mixing and culturing for 3 days. It can be seen from FIG3 that the cell number ratio of lung cancer organoid cells, lung cancer-associated fibroblasts, and vascular endothelial cells is appropriate, which is conducive to the spheroidization of the model and is more conducive to the construction of the tumor microenvironment. A lung cancer organoid model with a complex microvascular network and better simulation can be grown.

[0168] (2) Drug testing:

[0169] (1) Prepare appropriate concentrations of drug-containing culture medium: cisplatin + paclitaxel (cisplatin 6 μM, paclitaxel 4 μM), pemetrexed (20 μM);

[0170] (2) Transferring the vascularized lung cancer organoid spheroid model cultured in Example 1 to a 96-well plate;

[0171] (3) Observe and record the growth status of the vascularized lung cancer organoid spheroid model. Remove the original culture medium in the 96-well plate, add 100 μL of drug-containing culture medium to each well, and place it in an incubator at 37°C and 5% CO2 for culture.

[0172] (4) Observe and record the growth status of the vascularized lung cancer organoid spheroid model on day 3;

[0173] (5) On day 5, the growth status of the vascularized lung cancer organoid spheroid model was observed and recorded, and activity was detected using a CTG detection kit;

[0174] Add 100 μL CTG detection reagent to each well, shake on a shaker for 5 minutes, and incubate at room temperature for 25 minutes;

[0175] (6) After incubation, the cells were detected using a chemiluminescence microplate reader.

[0176] Figure 4 shows the status of the vascularized lung cancer organoid spheroid model prepared in Example 1 of the present disclosure on the 3rd and 5th days of culture after adding drug-containing culture medium during the drug sensitivity test, wherein the culture medium in the control group is blank culture medium, and the blank culture medium is added with 1% DMSO on the basis of the above-mentioned mixed culture medium.

[0177] FIG5 is a graph showing the results of detecting the lysates of the three groups of experiments in FIG4 using a chemiluminescence microplate reader.

[0178] In combination with Figures 4 and 5, it can be seen that both cisplatin + paclitaxel (a combination drug) and pemetrexed have a certain vascular inhibitory effect on the vascularized lung cancer organoid spheroid model constructed in Example 1 of the present disclosure, and the combination drug of cisplatin + paclitaxel is more sensitive to this lung cancer organoid spheroid.

[0179] Example 2

[0180] (I) Preparation of a vascularized lung cancer organoid spheroid model (forming a vascularized lung cancer organoid spheroid model on a drug sensitivity chip)

[0181] Step 1: Select primary lung cancer organoids and add pre-chilled PBS to a culture plate, adding 1 mL of PBS to each well. Collect the cells into a centrifuge tube, refrigerate at 4°C for 15 minutes, and centrifuge (1000 rpm, 5 minutes). Remove the supernatant to obtain a lung cancer organoid pellet.

[0182] Step 2: Add Tryple E for 8 minutes to digest into single cells, then centrifuge (1000 rpm, 5 minutes). Remove the supernatant, add PBS, and centrifuge again (1000 rpm, 5 minutes) to remove excess enzyme solution. The precipitate is resuspended in mixed culture medium and the cells are counted to determine the cell concentration of the lung cancer organoid suspension.

[0183] Step 3: Human primary lung cancer-associated fibroblasts were obtained, digested into single cells, resuspended in mixed culture medium, and counted;

[0184] Step 4: Human umbilical vein endothelial cells (HUVECs) were obtained, digested into single cells, resuspended in mixed culture medium, and counted;

[0185] Step 5, the lung cancer organoid cells, primary human lung cancer-associated fibroblasts, and human umbilical vein endothelial cells were mixed at a cell number ratio of 1:0.75:0.25 (the total number of cells per well was 5000, the number of lung cancer organoid cells was 2500 cells / well, the number of primary human lung cancer-associated fibroblasts was 1875 cells / well, and the number of human umbilical vein endothelial cells was 625 cells / well) to obtain a mixed suspension;

[0186] Step 6: Add the mixed suspension to a low-adsorption U-shaped 96-well plate and centrifuge at 1000 rpm for 5 minutes;

[0187] Step 7: Place the cells in an incubator at 37°C and 5% CO2 for the first stage of sphere formation culture;

[0188] Step 8: After 2 days of culture, remove the original culture medium from the well plate, add 5 μL of Matrigel, and transfer the spheroids to the middle culture chamber of the drug sensitivity chip. After gelation, add 30 μL of mixed culture medium to the middle culture chamber of the drug sensitivity chip and 50 μL of mixed culture medium to each of the two side reservoirs. Place the drug sensitivity chip in a rocking perfusion instrument set to 3° / 120 min. Place the rocking perfusion instrument in an incubator at 37°C and 5% CO2 for the second stage of spheroidization culture, which lasts for 1 day.

[0189] The diameter of the vascularized lung cancer organoid spheroid model prepared in Example 2 is 400 μm.

[0190] (2) Drug testing:

[0191] (1) Prepare drug-containing culture medium with appropriate concentrations: osimertinib + bevacizumab (osimertinib 1 μM, bevacizumab 1 μg / mL), docetaxel (3 μM);

[0192] (2) Observe and record the growth status of the vascularized lung cancer organoid spheroid model in the culture chamber of the drug sensitivity chip, and remove the original culture medium in the culture chamber;

[0193] (3) Add 50 μL of drug-containing culture medium to each of the two side reservoirs of the drug sensitivity chip and add 30 μL of drug-containing culture medium to the middle culture chamber;

[0194] (4) Place the drug-sensitive chip after drug addition into a rocking perfusion instrument, set the conditions to 3° / 120 min, and place the rocking perfusion instrument into an incubator at 37°C and 5% CO2 for dynamic culture;

[0195] (5) On the third day, the growth status of the vascularized lung cancer organoid spheroid model was observed and recorded, and 30 μL of drug-containing culture medium was added to the middle culture chamber;

[0196] (6) On day 5, the growth status of the vascularized lung cancer organoid spheroid model was observed and recorded, and activity was detected using a CTG detection kit;

[0197] (7) Discard all drug-containing culture medium in the reservoirs on both sides and add 20 μL of CTG diluent (1:1 dilution of CTG reagent stock solution and mixed culture medium);

[0198] (8) Without discarding the drug-containing culture medium in the middle culture chamber, directly add 50 μL of CTG stock solution and mix thoroughly by pipetting;

[0199] (9) Shake on an orbital shaker for 60 minutes to fully lyse the globulomers;

[0200] (10) All lysates were taken out and placed in a 96-well plate with completely opaque sides and bottom, and detected using a chemiluminescence enzyme reader.

[0201] Figure 6 shows the state of the vascularized lung cancer organoid spheroid model prepared in Example 2 of the present disclosure on the 3rd and 5th days of culture after adding the drug-containing culture medium during the drug sensitivity test. Figure 7 is a graph showing the results of the detection of the lysates of the three groups of experiments in Figure 6 using a chemiluminescence microplate reader. Combining Figures 6 and 7, it can be seen that the spheroid model can grow abundant blood vessels by dynamic culture using a drug sensitivity chip. Osimertinib + bevacizumab (another mixed drug) has a significant vascular inhibitory effect on the vascularized lung cancer organoid spheroid model constructed in Example 2 of the present disclosure, and docetaxel has no obvious vascular inhibitory effect on this sample. At the same time, the combination drug of osimertinib + bevacizumab is more sensitive to this sample.

[0202] Example 3

[0203] (I) Preparation of vascularized colorectal cancer organoid spheroid model

[0204] Step 1: Obtain primary colorectal cancer organoids. Add pre-chilled PBS to the culture well plate, add 1 mL of PBS to each well, collect the cells into a centrifuge tube, refrigerate at 4°C for 15 minutes, centrifuge (1000 rpm, 5 minutes), and remove the supernatant to obtain the colorectal cancer organoid pellet.

[0205] Step 2: Add Tryple E for 8 minutes to digest into single cells, then centrifuge (1000 rpm, 5 minutes). Remove the supernatant, add PBS, and centrifuge again (1000 rpm, 5 minutes) to remove excess enzyme solution. Obtain a precipitate, add mixed culture medium, and count the cells to determine the cell concentration of the colorectal cancer organoid suspension.

[0206] In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:0.8;

[0207] Step 3: Obtain primary human colorectal cancer-associated fibroblasts, digest them into single cells, resuspend them in the above-mentioned mixed culture medium, and count them;

[0208] Step 4: Human umbilical vein endothelial cells (HUVECs) were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0209] Step 5: Mix colorectal cancer organoid cells, primary human colorectal cancer-associated fibroblasts, and human umbilical vein endothelial cells at a cell number ratio of 34:5:8 (the total number of cells per well is 5000, the number of colorectal cancer organoid cells is 3617 cells / well, the number of primary human colorectal cancer-associated fibroblasts is 532 cells / well, and the number of human umbilical vein endothelial cells is 851 cells / well) to obtain a colorectal cancer organoid multicellular suspension;

[0210] Step 6: Add the colorectal cancer organoid multicellular suspension into a low-adhesion U-shaped 96-well plate and centrifuge at 1000 rpm for 5 minutes.

[0211] Step 7: Place the cells in an incubator at 37°C and 5% CO2 for the first stage of sphere formation culture;

[0212] Step 8. After 2 days of culture, the original culture medium in the well plate was aspirated, 5 μL of hydrogel was added, and the spheroids were transferred to the middle culture chamber of the drug sensitivity chip. After gelation, 30 μL of mixed culture medium was added to the middle culture chamber of the drug sensitivity chip, and 50 μL of mixed culture medium was added to each of the liquid reservoirs on both sides. The drug sensitivity chip was placed in a rocking perfusion instrument set to 3° / 120 min. The rocking perfusion instrument was placed in an incubator at 37°C and 5% CO2 for the second stage of spheroid culture. The culture time was 1 day. The prepared vascularized colorectal cancer organoid spheroid model had a diameter of 400 μm.

[0213] (2) Drug sensitivity test:

[0214] (1) Prepare a medium containing SN-38 (100 nM) at an appropriate concentration.

[0215] (2) Observe and record the growth status of the vascularized colorectal cancer organoid spheroid model in the culture chamber of the drug sensitivity chip, and remove the culture medium in the culture chamber;

[0216] (3) Add 50 μL of drug-containing culture medium to each of the two side reservoirs of the drug sensitivity chip and add 30 μL of drug-containing culture medium to the middle culture chamber;

[0217] (4) Place the drug-sensitive chip after drug addition into a rocking perfusion instrument, set the conditions to 3° / 120 min, and place the rocking perfusion instrument into an incubator at 37°C and 5% CO2 for dynamic culture;

[0218] (5) On the third day, the growth status of the vascularized colorectal cancer organoid spheroid model was observed and recorded, and 30 μL of drug-containing culture medium was added to the middle culture chamber;

[0219] (6) On day 5, the growth status of the vascularized colorectal cancer organoid spheroid model was observed and recorded, and activity was detected using a CTG detection kit;

[0220] (7) Discard all drug-containing culture medium in the reservoirs on both sides and add 20 μL of CTG diluent (1:1 dilution of CTG reagent stock solution and mixed culture medium);

[0221] (8) Without discarding the drug-containing culture medium in the middle culture chamber, directly add 50 μL of CTG stock solution and mix thoroughly by pipetting;

[0222] (9) Shake on a shaker for 5 minutes and incubate at room temperature for 25 minutes;

[0223] (10) All lysates were taken out and placed in a 96-well plate with completely opaque sides and bottom, and detected using a chemiluminescence enzyme reader.

[0224] Figure 8 shows the status of the vascularized colorectal cancer organoid spheroid model prepared in Example 3 of the present disclosure on days 3 and 5 of culture after adding drug-containing culture medium during drug sensitivity testing. Figure 9 shows the results of testing the lysate in Figure 8 using a chemiluminescence microplate reader. Combining Figures 8 and 9, it can be seen that the inhibition rate of SN-38 on the cell activity of this colorectal cancer organoid spheroid is 17%, and the inhibitory effect is not significant. Here, control refers to the cell activity of the control group (the same below).

[0225] Example 4

[0226] (I) Preparation of vascularized pancreatic cancer organoid spheroid model

[0227] Step 1: Obtain primary pancreatic cancer organoids. Add 1 mL of pre-chilled PBS to each well of a culture plate. Collect the cells into a centrifuge tube, refrigerate at 4°C for 15 minutes, and centrifuge (1000 rpm for 5 minutes). Remove the supernatant to obtain a pancreatic cancer organoid pellet.

[0228] Step 2: Add Tryple E for 8 minutes to digest into single cells, then centrifuge (1000 rpm, 5 minutes). Remove the supernatant, add PBS, and centrifuge again (1000 rpm, 5 minutes) to remove excess enzyme solution. Obtain a precipitate, add mixed culture medium, and count the cells to determine the cell concentration of the pancreatic cancer organoid suspension.

[0229] In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:1.2;

[0230] Step 3: Immortalized human pancreatic cancer-associated fibroblasts were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0231] Step 4: Human umbilical vein endothelial cells (HUVECs) were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0232] Step 5: Pancreatic cancer organoid cells, human pancreatic cancer-associated fibroblast immortalized cells, and human umbilical vein endothelial cells are mixed at a cell number ratio of 2:1:1 (the total number of cells per well is 5000, the number of pancreatic cancer organoid cells is 2500 cells / well, the number of human pancreatic cancer-associated fibroblast immortalized cells is 1250 cells / well, and the number of human umbilical vein endothelial cells is 1250 cells / well) to obtain a pancreatic cancer organoid multicellular suspension;

[0233] Step 6: Add the pancreatic cancer organoid multicellular suspension into a low-adhesion U-shaped 96-well plate and centrifuge at 1000 rpm for 5 minutes.

[0234] Step 7: Place the cells in an incubator at 37°C and 5% CO2 for the first stage of sphere formation culture;

[0235] Step 8. After 2 days of culture, the original culture medium in the well plate was aspirated, 5 μL of hydrogel was added, and the spheroids were transferred to the middle culture chamber of the drug sensitivity chip. After gelation, 30 μL of mixed culture medium was added to the middle culture chamber of the drug sensitivity chip, and 50 μL of mixed culture medium was added to the liquid reservoirs on both sides. The drug sensitivity chip was placed in a rocking perfusion instrument set to 3° / 120 min. The rocking perfusion instrument was placed in an incubator at 37°C and 5% CO2 for the second stage of spheroid culture. The culture time was 1 day. The diameter of the prepared vascularized pancreatic cancer organoid spheroid model was 400 μm.

[0236] (2) Drug sensitivity test:

[0237] (1) Prepare medium containing drug at an appropriate concentration: gemcitabine (10 μM);

[0238] (2) Observe and record the growth status of the vascularized pancreatic cancer organoid spheroid model in the culture chamber of the drug sensitivity chip, and remove the culture medium in the culture chamber;

[0239] (3) Add 50 μL of drug-containing culture medium to each of the two side reservoirs of the drug sensitivity chip and add 30 μL of drug-containing culture medium to the middle culture chamber;

[0240] (4) Place the drug-sensitive chip after drug addition into a rocking perfusion instrument, set the conditions to 3° / 120 min, and place the rocking perfusion instrument into an incubator at 37°C and 5% CO2 for dynamic culture;

[0241] (5) On the third day, the growth status of the vascularized pancreatic cancer organoid spheroid model was observed and recorded, and 30 μL of drug-containing culture medium was added to the middle culture chamber;

[0242] (6) On day 5, the growth status of the vascularized pancreatic cancer organoid spheroid model was observed and recorded, and the activity was detected using a CTG detection kit;

[0243] (7) Discard all drug-containing culture medium in the reservoirs on both sides and add 20 μL of CTG diluent (1:1 dilution of CTG reagent stock solution and mixed culture medium);

[0244] (8) Without discarding the drug-containing culture medium in the middle culture chamber, directly add 50 μL of CTG stock solution and mix thoroughly by pipetting;

[0245] (9) Shake on a shaker for 5 minutes and incubate at room temperature for 25 minutes;

[0246] (10) All lysates were taken out and placed in a 96-well plate with completely opaque sides and bottom, and detected using a chemiluminescence enzyme reader.

[0247] Figure 10 shows the vascularized pancreatic cancer organoid spheroid model prepared in Example 4 of the present disclosure, after adding drug-containing culture medium during drug sensitivity testing on days 3 and 5 of culture. Figure 11 shows the results of chemiluminescence microplate reader analysis of the lysate in Figure 10 . Combining Figures 10 and 11, it can be seen that gemcitabine inhibits cell viability in this pancreatic cancer organoid spheroid by 40%, but gemcitabine has no significant inhibitory effect on vascular growth.

[0248] Example 5

[0249] Preparation of a vascularized liver organoid spheroid model

[0250] Step 1: Obtain primary liver organoids. Add 1 mL of pre-chilled PBS to each well of the culture plate. Collect the cells into centrifuge tubes, refrigerate at 4°C for 15 minutes, and centrifuge (1000 rpm for 5 minutes). Remove the supernatant to obtain the liver organoid pellet.

[0251] Step 2: Add Tryple E for 8 minutes to digest into single cells, then centrifuge (1000 rpm, 5 minutes). Remove the supernatant, add PBS, and centrifuge again (1000 rpm, 5 minutes) to remove excess enzyme solution. Obtain a precipitate, add mixed culture medium, and count the cells to determine the cell concentration of the liver organoid suspension.

[0252] In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:0.8;

[0253] Step 3: Primary human liver-associated fibroblasts were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0254] Step 4: Human umbilical vein endothelial cells (HUVECs) were obtained, digested into single cells, resuspended in the above-mentioned mixed culture medium, and counted;

[0255] Step 5, the liver organoid cells, primary human liver-associated fibroblasts, and human umbilical vein endothelial cells were mixed at a cell number ratio of 2:3:3 (the total number of cells per well was 5000, the number of liver organoid cells was 1250 cells / well, the number of primary human liver fibroblasts was 1875 cells / well, and the number of human umbilical vein endothelial cells was 1875 cells / well) to obtain a mixed suspension;

[0256] Step 6: Add the mixed suspension to a low-adsorption U-shaped 96-well plate and centrifuge at 1000 rpm for 5 minutes;

[0257] Step 7: Place the cells in an incubator at 37°C and 5% CO2 for the first stage of sphere formation culture;

[0258] Step 8. After 2 days of culture, aspirate the original culture medium in the well plate, add 5uL of hydrogel, transfer the spheroids to the middle well of the drug sensitivity chip, and add 30μL of mixed culture medium after gelation. Add 50μL of mixed culture medium to each well on both sides of the drug sensitivity chip. Place the drug sensitivity chip in a rocking perfusion instrument and set the conditions to 3° / 120min. Place the rocking perfusion instrument in an incubator at 37°C and 5% CO2 for the second stage of spheroid culture. The culture time is 1 day. The diameter of the prepared vascularized liver organoid spheroid model is 400μm.

[0259] Hepatotoxicity testing:

[0260] (1) Prepare medium containing drugs at appropriate concentrations: clozapine (2.4 μM) and troglitazone (6.1 μM);

[0261] (2) Observe and record the growth status of the vascularized liver organoid spheroid model in the culture chamber of the drug sensitivity chip, and remove the culture medium in the culture chamber;

[0262] (3) Add 50 μL of drug-containing culture medium to the liquid storage chambers on both sides of the drug sensitivity chip, and add 30 μL of drug-containing culture medium to the culture chamber in the middle;

[0263] (4) Place the drug-sensitive chip after drug addition into a rocking perfusion instrument, set the conditions to 3° / 120 min, and place the rocking perfusion instrument into an incubator at 37°C and 5% CO2 for dynamic culture;

[0264] (5) On the third day, the growth status of the vascularized liver organoid spheroid model was observed and recorded, and 30 μL of drug-containing culture medium was added to the middle culture chamber;

[0265] (6) On day 5, the growth status of the vascularized liver organoid spheroid model was observed and recorded, and activity was detected using a CTG detection kit;

[0266] (7) Discard all drug-containing culture medium in the reservoirs on both sides and add 20 μL of CTG diluent (1:1 dilution of CTG reagent stock solution and culture medium);

[0267] (8) Without discarding the drug-containing culture medium in the middle culture chamber, add 50 μL of CTG stock solution directly and mix thoroughly by pipetting;

[0268] (9) Shake on a shaker for 5 minutes and incubate at room temperature for 25 minutes;

[0269] (10) All lysates were taken out and placed in a 96-well plate with completely opaque sides and bottom, and detected using a chemiluminescence enzyme reader.

[0270] Figure 12 shows the status of the vascularized liver organoid spheroid model prepared in Example 5 of the present disclosure on the 3rd and 5th days of culture after adding the drug-containing culture medium during the hepatotoxicity test, where NC is the blank control group. Figure 13 shows the results of the lysates of the three experimental groups in Figure 12 being tested using a chemiluminescence microplate reader. Combining Figures 12 and 13, it can be seen that clozapine and troglitazone both cause a certain degree of damage to the liver, especially clozapine, which has a stronger toxic effect on the liver.

[0271] The drug sensitivity chip used in the present disclosure adopts, for example, the cell culture devices shown in application numbers CN2023105307566, CN2023211335836, CN2023105302350, and CN202310530706.8. For example, taking the cell culture device shown in application number CN202310530706.8 as an example, referring to FIG14 , the middle culture chamber of the drug sensitivity chip described in the present disclosure refers to the culture chamber 10a in the cell culture device, and the liquid storage chambers on both sides of the drug sensitivity chip described in the present disclosure refer to the liquid storage chamber 10b in the cell culture device. There are two liquid storage chambers 10b. In the flow direction from the liquid storage chamber 10b to the culture chamber 10a, the two liquid storage chambers 10b are symmetrically arranged, and the culture chamber 10a is located between the two liquid storage chambers 10b. With this setting, the cell culture device can be swung. At this time, during the swing, the culture medium can move back and forth between the two liquid storage chambers 10b, and dynamic culture can be achieved by generating physical and chemical stimuli such as fluid shear force, mechanical stress, and biochemical concentration gradient. While showing more realistic physiological functions, it can better realize the material exchange between the culture medium and the biological sample, and can take away the excretions of the biological sample, which can better simulate the real cell environment.

[0272] The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A method for constructing a vascularized organoid spheroid model, characterized in that: The following steps are involved: S100: preparing or obtaining an organoid, wherein the organoid is a primary organoid or a passaged organoid, and digesting the primary organoid or the passaged organoid to obtain an organoid cell; Obtain fibroblasts and vascular endothelial cells; S200: mixing the organoid cells, the fibroblasts and the vascular endothelial cells, and resuspending them in a culture medium to obtain an organoid multicellular suspension; S300: inoculating the organoid multicellular suspension into a culture chamber or a culture well for spheroid culture to form the vascularized organoid spheroid model through self-assembly.

2. The construction method according to claim 1, characterized in that: The organoid is a primary tumor organoid or a passaged tumor organoid; Digesting the primary tumor organoid or the passaged tumor organoid into single cells to obtain tumor organoid cells; The fibroblasts include tumor-associated fibroblasts; The cell number ratio of the tumor organoid cells, the tumor-associated fibroblasts and the vascular endothelial cells is 1:(0.1-1):(0.05-1).

3. The construction method according to claim 1, characterized in that: The culture medium is a mixed culture medium; The mixed culture medium at least includes a first culture medium for culturing the organoid cells and a second culture medium for culturing the vascular endothelial cells; In the mixed culture medium, the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2).

4. The construction method according to claim 3, characterized in that: In step S300, when the organoid multicellular suspension is inoculated into a culture chamber, the culture chamber has a U-shaped bottom; or, When the organoid multicellular suspension is inoculated into a culture well, the culture well has a U-shaped bottom.

5. The construction method according to claim 4, characterized in that: The spheroidization culture includes a first stage spheroidization culture and a second stage spheroidization culture; The first stage of sphere formation culture includes: inoculating the organoid multicellular suspension into the culture chamber or the culture well for agglomeration treatment to make the cells in the organoid multicellular suspension agglomerate, and then placing it in a culture environment for culture for 1 to 5 days to obtain an organoid sphere aggregate model intermediate with clear boundaries and microvessels, and then performing the second stage of sphere formation culture.

6. The construction method according to claim 5, characterized in that: The conditions for the second stage of spheroid culture include: discarding the original culture medium in the culture cavity or the culture well containing the organoid spheroid model intermediate, adding cell culture scaffold material, adding the mixed culture medium after gelation, and placing in a culture environment for culture for 1 to 2 days.

7. The construction method according to claim 5, characterized in that: The conditions of the second stage of spheroid culture include: discarding the original culture medium in the culture chamber or the culture well containing the organoid spheroid model intermediate, adding cell culture scaffold material, transferring to the culture chamber or culture well of the drug sensitivity chip, adding the mixed culture medium after gelation, placing in the culture environment for dynamic culture, and the culture time is 1 to 2 days; The drug sensitivity chip culture chamber and the drug sensitivity chip culture hole have a U-shaped bottom and are interconnected.

8. The construction method according to claim 7, characterized in that: The conditions of the dynamic culture include: The flow perfusion culture is performed in the culture environment with a rocking speed of 3° to 10° for 15 min to 120 min.

9. The construction method according to claim 2, characterized in that: The organoid is a primary lung cancer organoid, an iPS stem cell differentiated lung cancer organoid, or a passaged lung cancer organoid; Digesting the primary lung cancer organoid, the iPS stem cell differentiated lung cancer organoid, or the passaged lung cancer organoid into single cells to obtain lung cancer organoid cells; The fibroblasts include lung cancer associated fibroblasts; The cell number ratio of the lung cancer organoid cells, the lung cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.4-0.8):(0.1-0.5); Preferably, the total number of the lung cancer organoid cells, the fibroblasts and the vascular endothelial cells is 5000 to 2*10 4 indivual.

10. The construction method according to claim 2, characterized in that: The organoid is a primary colorectal cancer organoid or a passaged colorectal cancer organoid; Digesting the primary colorectal cancer organoid or the passaged colorectal cancer organoid into single cells to obtain colorectal cancer organoid cells; The fibroblasts include colorectal cancer-associated fibroblasts; The cell number ratio of the colorectal cancer organoid cells, the colorectal cancer-associated fibroblasts and the vascular endothelial cells is 1:(0.1-0.8):(0.2-0.6).

11. The construction method according to claim 2, characterized in that: The organoid is a primary pancreatic cancer organoid or a passaged pancreatic cancer organoid; Digesting the primary pancreatic cancer organoid or the passaged pancreatic cancer organoid into single cells to obtain pancreatic cancer organoid cells; The fibroblasts include pancreatic cancer-associated fibroblasts; The cell number ratio of the pancreatic cancer organoid cells, the pancreatic cancer-associated fibroblasts and the vascular endothelial cells is 1:(0.2-0.7):(0.2-0.7).

12. The construction method according to claim 1, characterized in that: The organoid is a primary non-tumor organoid or a passaged non-tumor organoid; Digesting the primary non-tumor organoid or the passaged non-tumor organoid into single cells to obtain non-tumor organoid cells; The non-tumor organoid is selected from any one of a liver organoid, a lung organoid, a colorectal organoid or a pancreatic organoid; The fibroblasts include related fibroblasts.

13. A vascularized organoid spheroid model, characterized in that: The vascularized organoid spheroid model has a spheroid structure, in which a plurality of organoids are aggregated and grown; The vascularized organoid spheroid model has microvessels growing from the inside out; The vascularized organoid spheroid model also includes an organoid microenvironment composed of fibroblast structures and microvessels; The diameter of the vascularized organoid spheroid model ranges from 250 μm to 800 μm.

14. The vascularized organoid spheroid model according to claim 13, characterized in that: The vascularized organoid spheroid model has cell proliferation, and the degree of cell proliferation increases gradually from the inside to the outside; The oxygen content of the vascularized organoid spheroid model gradually increases from the inside to the outside.

15. The vascularized organoid spheroid model according to claim 13, characterized in that: The vascularized organoid spheroid model is constructed according to the construction method according to any one of claims 1 to 12.

16. Use of the vascularized organoid spheroid model according to any one of claims 13 to 15 in detection; The detection includes any one of drug screening, drug sensitivity detection, drug toxicity detection or non-drug substance toxicity detection.

17. The use according to claim 16, characterized in that The application comprises at least the following steps: Adding the drug to be tested into the culture chamber or culture well containing the vascularized organoid spheroid model, culturing in a culture environment for 3 to 5 days, and detecting changes in indicators of the vascularized organoid spheroid model; or, Adding the drug to be tested into the culture chamber or culture well of the drug sensitivity chip containing the vascularized organoid spheroid model, placing it in a culture environment for dynamic culture for 3 to 5 days, and detecting changes in indicators of the vascularized organoid spheroid model; The indicator changes include at least one of the changes in shape, size, activity, vascular morphology, protein molecule expression or distribution of the vascularized organoid spheroid model.

18. A drug detection method, characterized in that: The following steps are involved: S100: preparing or obtaining organoids, wherein the organoids are digested to obtain organoid cells, fibroblasts and vascular endothelial cells; S200: mixing the organoid cells, the fibroblasts and the vascular endothelial cells, and resuspending them in a culture medium to obtain an organoid multicellular suspension; S300: inoculating the organoid multicellular suspension into a culture chamber or a culture well for spheroid culture to form the vascularized organoid spheroid model through self-assembly; S400: adding a drug to be tested into a culture chamber or a culture well containing the vascularized organoid spheroid model, culturing the model in a culture environment, and then detecting changes in indicators of the vascularized organoid spheroid model; Preferably, the organoid is as described in any one of claims 1-2 and 9-12; the fibroblasts include tumor-associated fibroblasts; the cell number ratio of the tumor organoid cells, the tumor-associated fibroblasts and the vascular endothelial cells is 1:(0.1-1):(0.05-1); Preferably, the spheroid culture is as described in any one of claims 5-8.

19. A culture medium composition, characterized in that: The culture medium composition comprises a first culture medium for culturing organoid cells and a second culture medium for culturing vascular endothelial cells, and the mass ratio of the first culture medium to the second culture medium is 1:(0.5-2); Preferably, the culture medium composition is used for the construction method according to any one of claims 1 to 12, or for the construction of the vascularized organoid spheroid model according to any one of claims 13 to 15; Preferably, the mass ratio of the first culture medium to the second culture medium is 1:(0.8-1.2); Preferably, the organoid cell is as described in any one of claims 1-2 and 9-12.

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