Printing method and equipment for microenvironment containing organoids based on 3D printing

Abstract

The invention provides a 3D printing-based printing method and equipment for a microenvironment containing organoids, and relates to the technical field of organoids. The method comprises the following steps: acquiring to-be-printed organoid cell suspension and microenvironment cell suspension; placing any suspension in a spray head of 3D printing equipment, determining a first printing position of the suspension on a substrate through a microscope in the printing equipment, spraying the suspension onto a PDMS chip in the printing equipment by the spray head, and printing to obtain organoids or microenvironment cells; and placing the other suspension in a spray head of 3D printing equipment, determining a second printing position of the other suspension on the substrate through a microscope in the printing equipment, spraying the other suspension onto the PDMS chip in the printing equipment by the spray head, and printing to obtain the microenvironment containing the organoid. The 3D printing technology is used for accurately simulating the form of original tissue and the constitution of tumor cells and stromal cells of genome characteristics, and the interaction relation between the tumor cells and the stromal cells is researched.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and more specifically, to a printing method, device, medium, and program product for a microenvironment containing organoids based on 3D printing. Background Art

[0002] Organoids are cell clusters formed through cultivation that have a certain morphological structure and function and are similar to organs. The cultivation of organoids realizes the high-information complementarity of existing 2D cultivation methods and animal model systems. The difference of organoids is that cells first grow in basement membrane gels and then develop into a few 3D-shaped cell cultures. Tumor organoids are breakthrough frontier technologies emerging under the development of precision medicine, referring to in vitro three-dimensional models constructed from patient-derived tumors under specific culture conditions. It retains the characteristics of the tumor in the patient's body to a great extent and is a preclinical model with great potential. However, the lack of a tumor microenvironment is currently the main technical bottleneck of tumor organoids.

[0003] Lung cancer is a malignant tumor with a high incidence and high mortality rate globally. Its easy occurrence of distant metastasis is one of the main reasons for the high mortality rate. In particular, non-small cell lung cancer (NSCLC) accounts for about 85% of the total number of lung cancers, and 30%-50% of these patients will develop brain metastases during the course of the disease. Therefore, the research on the metastasis of NSCLC has important clinical significance and urgency. At present, although certain progress has been made in the targeted therapy for NSCLC, due to the relatively low operable mutation rate in some patients, not all patients can benefit from targeted therapy. In addition, some targeted anti-cancer drugs also have non-target effects, making the treatment effect unsatisfactory. Therefore, it is necessary to establish a preclinical model composed of tumor cells and stromal cells that can accurately simulate the morphological and genomic characteristics of the original tissue, study the interaction relationship between them, and provide a reliable basis for the formulation of clinical treatment plans. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a printing method, device, medium, and program product for a microenvironment containing organoids based on 3D printing; the method of the present invention can accurately simulate the morphological and genomic characteristics of the original tissue by 3D printing technology, study the interaction relationship between tumor cells and stromal cells, so as to provide a reliable basis for the formulation of clinical treatment plans.

[0005] The first aspect of the present application discloses a printing method for a microenvironment containing organoids based on 3D printing, and the method includes:

[0006] S1: Obtain the organoid cell suspension and the microenvironment cell suspension to be printed;

[0007] S2: Place the first suspension in the nozzle of a 3D printing device. Determine the first printing position of the first suspension on the substrate through the microscope in the printing device. The nozzle sprays the first suspension onto the PDMS chip in the printing device to print an organoid or microenvironment cells; the first suspension is either the organoid or the microenvironment cell suspension;

[0008] S3: Place the second suspension in the nozzle of the 3D printing device. Determine the second printing position of the second suspension on the substrate through the microscope in the printing device. The nozzle sprays the second suspension onto the PDMS chip in the printing device to print a microenvironment containing organoids; the second suspension is either the microenvironment or the organoid cell suspension, and the first suspension and the second suspension are different.

[0009] In some embodiments, when determining the second printing position in S3, determine the distance between the second printing position and the first printing position according to the developmental stage of the tumor cells, and determine the second printing position based on the first printing position and the distance;

[0010] Alternatively, when determining the second printing position in S3, determine the distance between the second printing position and the first printing position according to the research purpose, and determine the second printing position based on the first printing position and the distance;

[0011] Optionally, the first printing position and the second printing position are arranged at coordinate points on the micron or millimeter scale;

[0012] Optionally, a substrate is laid on the bottom layer of the PDMS chip;

[0013] Optionally, the distance between the nozzle and the substrate is less than 0.3 cm.

[0014] In some embodiments, the method further includes: real-time monitoring of the state of the organoid cells invading the microenvironment cells in the microenvironment cell suspension in the organoid cell suspension by live cell fluorescence tracing technology under a microscope, and adjusting the composition and conditions of the cell culture medium according to the state.

[0015] In some embodiments, after S3, the method further includes S4: crosslink the microenvironment cells containing organoids on the PDMS chip and place them in a cell culture medium for culture to obtain a 3D cell precipitate, and this 3D cell precipitate is the research model;

[0016] Optionally, the crosslinking method includes: placing the PDMS chip in a dish and crosslinking for a first period of time at a first temperature;

[0017] Optionally, the diameter of the dish is 50 cm;

[0018] Optionally, the first temperature is 37 °C;

[0019] Optionally, the first time period is 45 min.

[0020] In some embodiments, the method for preparing the organoid cell suspension includes: S101-1: obtaining an organoid sample of a tumor; S101-2: washing the organoid sample with PBS, removing the surface serum, adding TrypLE and pipetting evenly, placing it in an incubator for digestion and neutralizing with a neutralizing solution, centrifuging the neutralized neutralizing solution (transferring it to a centrifuge tube pre-rinsed with PBS for centrifugation; S101-3: adding TrypLE to the precipitate of the neutralized neutralizing solution in S101-2, placing it in an incubator for digestion, and neutralizing with a neutralizing solution, centrifuging the neutralized neutralizing solution; S101-4: resuspending the precipitate of the neutralized neutralizing solution in S101-3, and the resuspended liquid is the organoid cell suspension; optionally, the density of the organoids in the resuspended liquid is greater than or equal to 4×10^4 cells / ml.

[0021] In some embodiments, the method for preparing the microenvironment cell suspension includes: S102-1: washing the microenvironment cells with PBS; S102-2: submerging the microenvironment cells with trypsin, placing them in an incubator for digestion, and then neutralizing and centrifuging with the corresponding medium for the microenvironment cells; S102-3: resuspending the precipitate after centrifugation in S102-2 to obtain the microenvironment cell suspension; optionally, the microenvironment cells include: H1299, HFL1.

[0022] In some embodiments, the PDMS chip is provided with a flow channel and a plurality of spray holes communicating with the flow channel, and the cell suspension flows through the flow channel to the spray holes; the chip is moved along a preset path to sequentially eject the cell suspension in a plurality of spray hole groups to a specified position on the surface of the cell culture device to form printed cell clusters, and one spray hole group includes one or more of the spray holes.

[0023] The second aspect of the present application discloses a computer device, characterized in that the device includes: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the above method.

[0024] The third aspect of the present application discloses a computer-readable storage medium, characterized in that a computer program is stored thereon, and when the computer program is executed by a processor, the steps of the above method are implemented.

[0025] The fourth aspect of the present application discloses a computer program product, including a computer program, characterized in that when the computer program is executed by a processor, the steps of the above method are implemented.

[0026] The present application has the following beneficial effects:

[0027] 1. The present application innovatively discloses a printing method for a microenvironment containing organoids. This method sequentially and purposefully prints organoids and microenvironment cells through a 3D printing device to construct a preclinical model composed of tumor cells and stromal cells that can accurately simulate the morphological and genomic characteristics of the original tissue. This model has organoids and stromal cells with high survival rates (through 3D printing technology, the survival rates of organoids and stromal cells within seven days are higher than 95%), can maintain the 3D morphology of cells (it is proved that different types of cells can still maintain their 3D morphology within seven days, which is an important achievement under 3D inkjet printing conditions; all cells and lung cancer organoids grow in a highly active 3D form under 3D inkjet printing, which is completely different from the traditional cell culture mode. This 3D growth form is closer to the in vivo physiological conditions and provides a more real and reliable simulation platform for cell research. Compared with traditional 2D culture, the highly active 3D growth form can better maintain the functions and phenotypes of cells and provide more accurate results for disease research and drug screening.), has strong bionic advantages (under 3D co-culture, it is observed that the organoids and fibroblasts, and tumor cells and fibroblasts show a wrapping phenomenon, which indicates that this technology can promote the interaction between cells and the formation of tissue morphology, which reflects that this model has more bionic advantages in constructing the microenvironment of lung cancer brain metastasis). This model can study the interaction relationships between each other to predict the clinical results of targeted therapy and provide a reliable basis for the formulation of clinical treatment plans.

[0028] 2. Based on inkjet printing technology, the present application can arrange organoids highly actively at precise coordinate points. By determining the first printing position and the second printing position, the customization of the positional relationship between lung cancer organoids and microenvironment stromal cells can be achieved; in addition, through the printing method disclosed in the present application, it can also ensure that organoids and stromal cells grow three-dimensionally on the same plane, thereby achieving an accurate simulation of lung cancer and its tumor microenvironment. It solves the following problems existing in the prior art one by one: ① Currently, the prior art cannot ensure the arrangement of highly active organoids at precise coordinate points. ② It cannot achieve the patterned customization of the positional relationship between organoids and their tumor microenvironment stromal cells. ③ Traditional printing technologies, such as extrusion printing, cannot achieve the three-dimensional growth of cells on a single plane and simultaneously ensure the accurate positional relationship between multiple cells.

[0029] 3. The method disclosed in the present application can precisely customize the positional relationship between organoids and stromal cells, thereby more precisely ensuring the interaction distance and spatial positional relationship between different cells. This precision enables a better simulation of complex disease conditions, such as lung cancer brain metastasis, and a simulation closer to the physiological situation under bionic experimental conditions.

[0030] 4. This application uses 3D thermal bubble-based bioprinting. After the organoids and microenvironment cells form spheres at specific coordinates respectively, through daily observation of live cell tracers or observation of the expression levels of marker proteins by cellular immunofluorescence, relative changes in cell positions in the co-cultured tumor microenvironment system can be observed, which cannot be achieved by previous technical routes. Moreover, due to the particularity of 3D thermal bubble printing, the samples can be used for stable and repeatable 3D modeling of cell clusters through confocal microscopy layer scanning, which also cannot be achieved under traditional co-culture in Matrigel. The method of this application can solve the following problems existing in the current mainstream methods for studying the tumor microenvironment (such as 3D Co-culture of Cancer-Associated Fibroblast with Oral Cancer Organoids): The above mainstream method is to centrifuge tumor organoids and then mix them with microenvironment cells and place them in Matrigel to form cell spheres. It cannot achieve spatial position adjustment, and the arrangement order of cells is also disordered. Most importantly, Matrigel has a strong limiting effect on cells, and cells cannot move freely in Matrigel, which results in the observed cell interactions not involving changes in position.

[0031] 5. By selecting different types of microenvironment cells, including fibroblasts, endothelial cells, macrophages, etc., precisely controlling the distance between them and tumor cells, and adjusting the culture medium components, the states of tumor cells at different developmental stages are simulated. Based on the inkjet printing technology under the microscope, the organoids can be highly actively arranged with micron-level precision at precise coordinate points. The dynamic regulation is mainly reflected in the real-time monitoring through the fluorescence tracing technology of living cells under the microscope, and at the same time, the components and conditions of the culture medium are dynamically adjusted during the cell culture process. For example, according to the different times when tumors invade fibroblasts, from the initial pure 1640 (tumor culture medium) to the medium configured with a 1:1 ratio of 1640 and F12k (fibroblast culture medium) when contacting HFL1, to meet the needs of cells at different developmental stages, thereby realizing the dynamic regulation of the cell microenvironment. Looking ahead, this method will provide a more powerful tool for more precise tumor research and personalized treatment, and promote medical research towards the simulation of more real and complex biological systems. Through the special inkjet printing technology under the microscope in this study, the organoids can be precisely arranged at the millimeter level at precise coordinate points, and the mutual contact position relationship between the microenvironment cells and tumor cells can be maintained. Through dynamic observation, we found the interactions between different microenvironment cells and organoids, especially the special wrapping behavior phenomenon between organoids and fibroblasts. This phenomenon was also verified in the immunohistochemical staining of clinical lung cancer surgical sample sections, indicating that this scheme can reproduce the unique connection between the microenvironment and organoids in the lung cancer microenvironment. This unique connection and precise control make this scheme not only technically innovative, but also able to more realistically simulate the lung cancer microenvironment, providing a powerful tool and platform for tumor research and personalized treatment. In addition, the distance between tumor cells and microenvironment cells can be flexibly adjusted according to specific research objectives to ensure the effectiveness and pertinence of experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 It is a schematic flowchart of the method provided in the first aspect of the embodiment of the present invention;

[0034] Figure 2 It is a schematic diagram of a printing system based on 3D printing and including a microenvironment containing organoids provided in the second aspect of the embodiment of the present invention;

[0035] Figure 3It is a schematic diagram of a computer device provided by an embodiment of the present invention;

[0036] Figure 4 It is a schematic diagram of the architecture of an exemplary computing device provided by an embodiment of the present invention;

[0037] Figure 5 It is a schematic diagram of a storage medium provided by an embodiment of the present invention;

[0038] Figure 6 It is a schematic diagram of an inkjet printer and a printing setting interface provided by an embodiment of the present invention; (A) This schematic diagram shows the printing interface of a 3D thermal bubble bio - printer view. Our team uses this interface to coordinate the printing substrate and then finely control the printing area. (B) It shows the situation of the chip in the dish after printing a single type of cell. The white dots within the red frame are the cell spheres aggregated after printing. (C) It shows the situation of using a 3D thermal bubble bio - printer to simultaneously print H1299 cells and HFL1 to construct a brain tumor microenvironment. The left figure shows the situation after printing as displayed by the printer camera, and the right figure shows the situation under the microscope after printing. (D) It shows the situation of using a 3D thermal bubble bio - printer to simultaneously print lung cancer organoids and HFL1 to construct a tumor microenvironment. The left figure shows the situation after printing as displayed by the printer camera, and the right figure shows the situation under an inverted microscope. The lung cancer organoids are within the red square.

[0039] Figure 7 It is a schematic diagram of the comparison between 3D printing and traditional inoculation of lung cancer organoids provided by an embodiment of the present invention: (A) Comparison of organoids under different culture methods. There is no difference in the morphology of organoids between the traditional in Matrigel culture method and the on Matrigel method using 3D printing. (B) Comparison of cells under an inverted microscope light microscope using two methods: a pipette and a 3D inkjet printer. The cells printed using a 3D inkjet printer are more aggregated. (C) Schematic diagram of the comparison of the spatial distribution of lung cancer organoids in droplets using a 3D inkjet printer and a pipette;

[0040] Figure 8 It is a schematic diagram of the research process and histological verification of lung cancer organoids provided by an embodiment of the present invention: (A) Describes the research process of lung cancer organoids. (B) Shows the histological characteristics of LCOs derived from advanced lung cancer under H&E staining, and IHC staining of adenocarcinoma - derived LCOs was performed using classical subtype markers TTF - 1 and NapsinA;

[0041] Figure 9 It is a schematic diagram of the cell viability verification of 3D - printed lung cancer organoids and microenvironment stromal cells provided by an embodiment of the present invention;

[0042] Figure 10Schematic diagram of the three-dimensional growth of lung cancer and the microenvironment and the expression of marker proteins in the 3D printed model under a confocal microscope provided by an embodiment of the present invention: (A) shows the fibroblast clusters printed by a 3D printer and the expression of the classical marker αSMA therein. (B) presents the perivascular cell clusters of cerebral blood vessels printed by a 3D printer and the expression of the classical marker PDGFRβ therein. (C) shows the BrM3 cell clusters printed by a 3D printer and the expression of the classical markers CK7 and TTF-1 therein. (D) compares the organoid situation at the early stage P1 under the traditional dome cultivation method and shows the expression of the classical marker CK7 and the classical marker αSMA of fibroblast clusters therein. (E) shows the classical marker CK7 of organoids and the classical marker αSMA of fibroblast clusters under 3D printing technology. (F) shows the expression of the classical marker CK7 of organoids and the classical marker αSMA of fibroblast clusters under the co-culture of 3D printed fibroblasts and organoids under an inverted fluorescence microscope;

[0043] Figure 11 Schematic diagram of the spatial distribution of personalized lung cancer and the microenvironment using 3D bioprinting provided by an embodiment of the present invention: By using 3D printing technology, two combinations of HFL1 and BrM3, and HFL1 and pericytes are printed respectively to achieve their distribution patterns of being far apart (A), adjacent (B), and fused (C) in space;

[0044] Figure 12 Schematic diagram of observing the fusion and interaction of lung cancer organoids and the tumor microenvironment under an inverted fluorescence microscope using a cell tracer provided by an embodiment of the present invention. Detailed implementation manners

[0045] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0046] In some processes described in the specification, claims, and the above-mentioned drawings of the present invention, a plurality of operations appear in a specific order, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or may be executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions such as "first" and "second" in this article are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, nor do they limit that "first" and "second" are of different types.

[0047] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0048] Figure 1 FIG. 4 is a schematic flowchart of a printing method for a 3D printing-based microenvironment containing organoids provided by an embodiment of the present invention. Specifically, the method includes the following steps:

[0049] S1: Obtain an organoid cell suspension and a microenvironment cell suspension to be printed;

[0050] In some embodiments, the preparation method of the organoid cell suspension includes: S101-1: Obtain an organoid sample of a tumor; S101-2: Wash the organoid sample with PBS (3 times per dome and then drain), remove the surface serum, add TrypLE (200 μl per well) and pipette evenly, place it in an incubator for digestion (place it in a 37 °C incubator for digestion for 10 minutes, and after digestion, add 200 μl of refrigerated 4 °C DMEM / F12 (containing 10% serum) to each well for neutralization), and neutralize with the neutralizing solution, centrifuge the neutralized solution (transfer it to a centrifuge tube pre-rinsed with PBS for centrifugation (300 g, centrifugation for 5 minutes)); S101-3: Add TrypLE to the precipitate of the neutralized solution after centrifugation in S101-2, place it in an incubator for digestion, and neutralize with the neutralizing solution, centrifuge the neutralized solution (after discarding the supernatant, add 2 ml of TrypLE to the precipitate, digest in a 37 °C incubator for 10 minutes. After digestion, add 2 ml of refrigerated 4 °C DMEM / F12 (containing 10% serum) for neutralization, and then place it in a centrifuge for centrifugation (300 g, centrifugation for 5 minutes)); S101-4: Resuspend the precipitate of the neutralized solution after centrifugation in S101-3 (after discarding the supernatant, add 100 μl of PBS to fully resuspend the cell suspension), and the resuspended liquid is the organoid cell suspension; the density of organoids in the resuspended liquid is greater than or equal to 4×10^4 cells / ml, and it is placed on the printer nozzle for printing.

[0051] In some embodiments, the method for preparing the microenvironment cell suspension includes: S102-1: washing the microenvironment cells with PBS (after washing 3 times, blotting dry); S102-2: submerging the microenvironment cells with trypsin (1 ml, fully covering the culture flask), placing it in an incubator for digestion (digesting in an incubator at 37 °C for 4 min), neutralizing with the corresponding medium for the microenvironment cells (subsequently neutralizing with the corresponding medium for the cell line) and centrifuging (putting the liquid into an EP tube, centrifuging (1000 RPM, centrifuging for 5 min)); S102-3: resuspending the precipitate after centrifugation in S102-2 (after discarding the supernatant, adjusting the cell density to 2×10^6 cells / ml, and then adding an appropriate volume of PBS to resuspend the cells), to obtain the microenvironment cell suspension; optionally, the microenvironment cells include: H1299, HFL1.

[0052] In some embodiments, the PDMS chip is provided with a flow channel and a plurality of spray holes communicated with the flow channel, and the cell suspension flows through the flow channel to the spray holes; moving the chip along a preset path to sequentially eject the cell suspension in a plurality of spray hole groups to a designated position on the surface of the cell culture device to form printed cell clusters, and one spray hole group includes one or more of the spray holes.

[0053] In some embodiments, the microenvironment cells include any one or several of the following: fibroblasts, endothelial cells, macrophages, etc.

[0054] S2: placing the first suspension in the nozzle of a 3D printing device, determining the first printing position of the first suspension on the substrate through a microscope in the printing device, and spraying the first suspension from the nozzle onto the PDMS chip in the printing device to print organoids or microenvironment cells; the first suspension is either an organoid and microenvironment cell suspension.

[0055] Optionally, a substrate is laid on the bottom layer of the PDMS chip;

[0056] S3: placing the second suspension in the nozzle of a 3D printing device, determining the second printing position of the second suspension on the substrate through a microscope in the printing device, and spraying the second suspension from the nozzle onto the PDMS chip in the printing device to print a microenvironment containing organoids; the second suspension is either a microenvironment and organoid cell suspension, and the first suspension and the second suspension are different.

[0057] In some embodiments, if the first suspension is an organoid cell suspension, the second suspension is a microenvironment cell suspension; if the first suspension is a microenvironment cell suspension, the second suspension is an organoid cell suspension.

[0058] In some embodiments, when determining the second printing position in S3, the distance between the second printing position and the first printing position is determined according to the developmental stage of the tumor cells, and the second printing position is determined based on the first printing position and the distance; alternatively, when determining the second printing position in S3, the distance between the second printing position and the first printing position is determined according to the research purpose, and the second printing position is determined based on the first printing position and the distance; optionally, if the research purpose is to study the cell behavior relationship of the interaction between tumor cells and microenvironment cells, the distance between the first printing position and the second printing position is close to zero, preferably zero; optionally, if the research purpose is to evaluate the effect of microenvironment cells on the migration ability of tumor cells, the distance range between the first printing position and the second printing position is: greater than 2 microns and less than 10 centimeters; according to the research purpose, the distance between tumor cells and microenvironment cells can be customized. If the research purpose is to deeply explore the cell behavior relationship of the interaction between tumor cells and microenvironment cells, it is recommended to set the distance between the two to be close to zero to ensure direct contact and precise interaction between cells. In addition, if the research purpose is to evaluate the effect of microenvironment cells on the migration ability of tumor cells, the distance between the two can be set to range from several microns to several centimeters. This setting of the distance range can better simulate the actual in vivo environment, provide more real and diverse experimental data, and thus more comprehensively understand the role of microenvironment cells in the migration process of tumor cells. In summary, the distance between tumor cells and microenvironment cells should be flexibly adjusted according to the specific research objective to ensure the effectiveness and pertinence of the experimental results.

[0059] Optionally, the first printing position and the second printing position are arranged at coordinate points in the micron or millimeter level;

[0060] In some embodiments, the method further includes: real-time monitoring of the state of the microenvironment cells in the organoid cell suspension invading the microenvironment cell suspension by live cell fluorescence tracing technology under a microscope, and adjusting the composition and conditions of the cell culture medium according to the state.

[0061] In some embodiments, after S3, the method further includes S4: crosslinking the microenvironment cells containing organoids on the PDMS chip and culturing them in a cell culture medium to obtain a 3D cell precipitate, and this 3D cell precipitate is the research model; in this application, the liquid used for printing is a PBS plus cell resuspension, but after printing and crosslinking, the resuspension forms a stable 3D cell precipitate after incubation on the matrix gel. Specifically, the 3D thermal bubble bioprinting technology precisely prints the organoid and microenvironment cell suspensions onto the PDMS chip to achieve three-dimensional growth and finally form 3D cell spheres. This process ensures that the cells can maintain high activity and 3D morphology after printing.

[0062] Optionally, the crosslinking method includes: placing the PDMS chip in a dish and crosslinking for a first period of time at a first temperature to increase the adhesion between cells and the substrate; crosslinking refers to the process of forming covalent or non-covalent bonds between polymer chains or within molecules through chemical reactions or physical interactions, thereby forming a three-dimensional network structure. This crosslinked structure endows the polymer material with stronger mechanical properties, thermal stability, and chemical resistance.

[0063] Optionally, the diameter of the dish is 50 cm; optionally, the first temperature is 37 °C; optionally, the first period of time is 45 min.

[0064] In some embodiments, after S4, the method further includes S5: placing the crosslinked mixture in a cell culture medium for culturing to obtain cell microspheres, and the cell microspheres are the research models.

[0065] The microenvironment refers to the microenvironment cell line. Primary cells are generally passaged about 10 times, and most cells senesce and die. However, a very small number of cells can survive the "crisis" and continue to be passaged. The surviving cells can generally be passaged to 40 - 50 generations, which is called a cell strain. When it reaches more than 50 generations, another "crisis" will occur. At this time, the genetic material of some cells has changed and has the characteristics of canceration, so they may be passaged indefinitely, which is called a cell line.

[0066] It is also considered that a cell line refers to the cell population propagated after the primary cell culture is successfully passaged for the first time. It also refers to cultured cells that can be continuously passaged for a long time. From this, the later finite cell line and infinite cell line are derived. Therefore, in a narrow sense, a cell line refers to cells that can be continuously passaged, and in a broad sense, it refers to cells that can be passaged.

[0067] PBS is the abbreviation of phosphate buffer saline, which is usually called "phosphate buffered saline" in Chinese. A more accurate translation should be "phosphate buffered saline", which is a buffer solution widely used in biochemical experiments. Usually, 0.9% NaCl is added to PB, and it has an osmotic pressure and pH value similar to human body fluids.

[0068] 3D bioprinting is an emerging and cutting-edge technology that integrates multiple disciplines such as medicine, biology, materials, and machinery. Its application scope in the medical field has transitioned from simply manufacturing simulated organ morphology models to constructing biocompatible tissue repair materials and then to printing relevant tissue models with biological activity. Currently, 3D bioprinting can achieve precise control of the spatial distribution of cells and the surrounding microenvironment, which is efficient and repeatable, and has great potential in the construction of complex tissues and organs.

[0069] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention. As Figure 3 shown, the device may include: one or more processors, and one or more memories; wherein, computer-readable code is stored in the memory, and when the computer-readable code is run by the one or more processors, the above-described method can be executed.

[0070] The processor in this embodiment may be an integrated circuit chip with signal processing capabilities. The above-mentioned processor may be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components. It can implement or execute the various methods, operations and logic block diagrams disclosed in the embodiments of the present disclosure. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc., and may be of the X86 architecture or the ARM architecture.

[0071] Generally speaking, various exemplary embodiments of the present disclosure may be implemented in hardware or special circuits, software, firmware, logic, or any combination thereof. Some aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that can be executed by a controller, a microprocessor or other computing devices. When aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flowcharts or using certain other graphical representations, it will be understood that the blocks, devices, systems, technologies or methods described herein may be implemented as non-limiting examples in hardware, software, firmware, special circuits or logic, general hardware or controllers or other computing devices, or certain combinations thereof.

[0072] For example, the method or device according to an embodiment of the present disclosure may also be implemented by means of Figure 4 the architecture of the computing device 3000 shown. As Figure 4 shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, an input / output component 3060, a hard disk 3070, etc. The storage device in the computing device 3000, such as the ROM 3030 or the hard disk 3070, may store various data or files used for the processing and / or communication of the method provided by the present disclosure and the program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 the architecture shown is only exemplary, and when implementing different devices, one or more components shown in the Figure 4 computing device may be omitted according to actual needs.

[0073] An embodiment of the present invention also provides a computer-readable storage medium, as Figure 5 shown, which is a schematic diagram of the storage medium provided by the embodiment of the present invention. Computer-readable instructions 4010 are stored on the computer storage medium 4020. When the computer-readable instructions 4010 are run by a processor, the methods according to the embodiments of the present disclosure described with reference to the above drawings can be executed. The computer-readable storage medium in the embodiments of the present disclosure may be a volatile memory or a non-volatile memory, or may include both volatile and non-volatile memories. The non-volatile memory may be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), or a flash memory. The volatile memory may be a random access memory (RAM), which is used as an external cache. By way of example but not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDR SDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memories of the methods described herein are intended to include, but are not limited to, these and any other suitable types of memories. It should be noted that the memories of the methods described herein are intended to include, but are not limited to, these and any other suitable types of memories.

[0074] An embodiment of the present disclosure also provides a computer program product or system, including a computer program, which implements the steps of the above method when executed by a processor.

[0075] In some embodiments, this embodiment also discloses a printing system for a 3D-printed microenvironment containing organoids, as Figure 2 shown. The system includes:

[0076] An acquisition module 201: configured to acquire an organoid cell suspension and a microenvironment cell suspension to be printed;

[0077] A first printing module 202: configured to place a first suspension in a nozzle of a 3D printing device, determine a first printing position of the first suspension on a substrate through a microscope in the printing device, and the nozzle sprays the first suspension onto a PDMS chip in the printing device to print an organoid or microenvironment cells; the first suspension is any one of the organoid and microenvironment cell suspensions;

[0078] Second printing module 203: used to place the second suspension in the nozzle of the 3D printing device, determine the second printing position of the second suspension on the substrate through the microscope in the printing device, and the nozzle sprays the second suspension onto the PDMS chip in the printing device to print a microenvironment containing organoids; the second suspension is either the microenvironment or the organoid cell suspension, and the first suspension and the second suspension are different.

[0079] In some embodiments, the system further includes:

[0080] Research model culture module: used to crosslink the cells in the microenvironment containing organoids on the PDMS chip and place them in a cell culture medium for culture to obtain a 3D cell precipitate, which is the research model.

[0081] Example 1 A method for 3D thermo-bubble bioprinting 3D organoids, including the following steps:

[0082] Step 1: Obtain a cell suspension of the tissue to be printed;

[0083] The method for preparing the cell suspension is as follows:

[0084] Select a subcultured lung cancer organoid sample, count under the microscope to ensure that the quantity meets the standard, wash each dome 3 times with PBS and then drain, remove the surface serum, add 200 ul of TrypLE per well and pipette evenly, place it in an incubator at 37 °C for digestion for 10 minutes. After digestion, add 200 ul of cold 4 °C DMEM / F12 (containing 10% serum) per well for neutralization, and then transfer it to a centrifuge tube pre-rinsed with PBS for centrifugation (300 g, centrifugation for 5 min).

[0085] After discarding the supernatant, add 2 ml of TrypLE to the precipitate and digest it in an incubator at 37 °C for 10 min. After digestion, add 2 ml of cold 4 °C DMEM / F12 (containing 10% serum) for neutralization, and then place it in a centrifuge for centrifugation (300 g, centrifugation for 5 min).

[0086] After discarding the supernatant, add 100 ul of PBS to fully resuspend the cell suspension

[0087] Step 2: Print the cell suspension at a specific position on the substrate through 3D thermo-bubble view printing to obtain 3D on Matrigel organoids;

[0088] The method for thermo-bubble view printing is as follows:

[0089] Load 100 μl of PBS containing lung cancer organoids into the print head. At room temperature, with the aid of the microscope built into the printer, after determining the specific position under the microscope, use the view printing method to spray the cells onto the PDMS chip with a Matrigel substrate laid on the bottom layer. The distance between the print head and the substrate is less than 0.3 cm. When using this printing method, the volume of the liquid ejected per double-sided printing is 15.4 nl, and double-sided printing is performed 6 times. After printing, place the chip in a 50 cm dish and put it in an oven at 37 °C for 45 minutes for cross-linking to increase the adhesion of the cells to the substrate. After cross-linking is completed, put the print head into an ultrasonic cleaner and clean it for 5 minutes. Then, slowly inject the corresponding organoid medium along the wall using a pipette, and change the medium every 3 days.

[0090] Example 2 A method for simultaneously printing H1299 cells and HFL1 to construct a tumor microenvironment by 3D thermal bubble bioprinting, comprising the following steps:

[0091] Step 1: Obtain a cell suspension of the tissue to be printed;

[0092] The preparation methods of H1299 and HFL1 cell suspensions are as follows:

[0093] Wash the HFL1 or H1299 in the T25 culture flask three times with PBS and then suck it dry.

[0094] Fully cover the culture flask with 1 ml of trypsin, place it in an incubator at 37 °C for digestion for 4 min, and then neutralize it with the corresponding medium of the cell line. Put the liquid into an EP tube and centrifuge (1000 RPM, centrifuge for 5 min).

[0095] After discarding the supernatant, adjust the cell density to 2×10^6 cells / ml, then add an appropriate volume of PBS to resuspend the cells, and put them on the print head of the printer for preparation for printing.

[0096] Step 2: Simultaneously print the H1299 and HFL1 cell suspensions at specific positions on the substrate by 3D thermal bubble view printing, and then a tumor microenvironment image can be obtained;

[0097] The thermal bubble view printing method is as follows:

[0098] Load 100 μl of PBS containing H1299 into the print head. At room temperature, with the aid of the microscope built into the printer, after determining the specific position under the microscope, use the view printing method to spray the cells onto the PDMS chip with a Matrigel substrate laid on the bottom layer.

[0099] Replace the print head, load HFL1 cells into the print head, and with the aid of the microscope built into the printer, after determining the positional relationship between the second type of cells and the first type of cells under the microscope, use the view printing method to spray the cells onto a PDMS chip with a Matrigel substrate laid on the bottom layer.

[0100] After printing is completed, place the chip in a 50 cm dish and put it in an oven at 37 °C for 45 minutes for crosslinking to increase the adhesion between the cells and the substrate. After crosslinking is completed, put the nozzle into an ultrasonic cleaner and clean it for 5 minutes. Then, slowly inject the organoid culture medium along the wall with a pipette gun, and change the culture medium every 3 days.

[0101] Example 3 A method for 3D thermo-bubble bioprinting to simultaneously print 3D organoids and HFL1, comprising the following steps:

[0102] Step 1: Obtain a cell suspension of the tissue to be printed;

[0103] The preparation method of the organoid cell suspension is as follows:

[0104] Select an HFL1 sample, count under a microscope to ensure that the quantity meets the standard. After washing each dome 3 times with PBS and then draining it, remove the surface serum, add 200 μl of TrypLE per well and pipette evenly, place it in an incubator at 37 °C for digestion for 10 minutes. After digestion is completed, add 200 μl of DMEM / F12 (containing 10% serum) at 4 °C that has been refrigerated to each well for neutralization, and then transfer it to a centrifuge tube that has been pre-rinsed with PBS for centrifugation (300 g, centrifuge for 5 min).

[0105] After discarding the supernatant, add 2 ml of TrypLE to the precipitate and digest it in an incubator at 37 °C for 10 min. After digestion is completed, add 2 ml of DMEM / F12 (containing 10% serum) at 4 °C that has been refrigerated for neutralization, and then place it in a centrifuge for centrifugation (300 g, centrifuge for 5 min).

[0106] After discarding the supernatant, add 100 μl of PBS to fully resuspend the cell suspension

[0107] The preparation method of the HFL1 cell suspension is as follows:

[0108] Wash the cell line in a T25 culture flask three times with PBS and then drain it. Use 1 ml of trypsin to fully cover the culture flask, place it in an incubator at 37 °C for digestion for 4 min, and then neutralize it with the corresponding medium of the cell line. Transfer the liquid into an EP tube and centrifuge (1000 RPM, centrifuge for 5 min). After discarding the supernatant, adjust the cell density to 2×10^6 cells / ml, then add an appropriate volume of PBS to resuspend the cells, and place them on the printer nozzle for printing

[0109] Step 2: Print the lung cancer organoid and HFL1 cell suspension at a specific position on the substrate by 3D thermal bubble printing to obtain a 3D organoid;

[0110] The printing method of thermal bubble view is as follows:

[0111] Load 100ul of PBS containing lung cancer organoids into the nozzle, and at room temperature, use the printer's built-in microscope to determine the specific position under the microscope, and then use the view printing method to spray the cells onto the PDMS chip with the Matrigel substrate on the bottom.

[0112] Replace the print head, load HFL1 cells in the print head, and use the microscope that comes with the printer to determine the positional relationship between the second type of cells and the first type of cells under the microscope, and then use the visual printing method to spray the cells onto the PDMS chip with the Matrigel substrate at the bottom.

[0113] After printing, place the chip in a 50cm dish and put it in a 37℃ oven for 45 minutes for crosslinking to increase the adhesion between cells and the substrate. Place the nozzle in an ultrasonic cleaner for 5 minutes. After crosslinking, use a pipette to slowly inject organoid culture medium along the wall. Change the culture medium every 3 days.

[0114] like Figure 6 The following is a schematic diagram of the inkjet printer and the print setting interface: (A) This schematic diagram shows the 3D thermal bubble bioprinter view printing interface. Our team used this interface to coordinate the printing substrate and finely control the printing area. (B) It shows the chip in the dish after the single cell printing is completed. The white dots in the red frame are the cell spheres aggregated after printing. (C) It shows the 3D thermal bubble bioprinter printing H1299 cells and HFL1 to construct a brain tumor microenvironment at the same time. The left picture shows the printer camera showing the situation after printing, and the right picture shows the situation under the microscope after printing. (D) It shows the 3D thermal bubble bioprinter printing lung cancer organoids and HFL1 to construct a tumor microenvironment at the same time. The left picture shows the printer camera showing the situation after printing, and the right picture shows the situation under an inverted microscope. The red box is the lung cancer organoid.

[0115] In this application, a printing framework was constructed using PDMS (polydimethylsiloxane), and 3D spheroid culture of different types of cells was achieved using 3D thermal bubble inkjet printing technology. This inkjet printing technology has the ability to precisely control the number of cells printed each time, with an average of about 30 cells ejected per time at the specified position. The PDMS chip we used can provide a non-adhesive culture environment for various cells, and the coating formed by matrigel prevents cell adhesion. The overall survival rate of the bioprinted cells is above 95%. This indicates that the model constructed based on this inkjet printing technology has the characteristics of high cell activity, strong simulation, high throughput, high speed, precise controllability, and reproducibility.

[0116] Among them, the relevant experimental methods are as follows:

[0117] Preparation method of PDMS chip: Prepare a PDMS chip with a circular chamber with a diameter of 1.5 mm, and then remove the PDMS plate from the template. The PDMS plate and the coverslip are treated with oxygen plasma. The two layers are finally bonded to form the entire 3D printing chip. Cover 150 ul of 70% concentration of matrigel in the chip chamber as a substrate to prevent cell adhesion. Immediately after incubating in an incubator at 7 degrees for 30 minutes, cell printing treatment is carried out.

[0118] Cell sample source: The human lung cancer cell line PC9 was obtained from the Chinese Academy of Medical Sciences (Beijing, China). BM-derived cells (PC9-BrM1, PC9-BrM2, and PC9-BrM3) were derived from the parental cell line PC9, and tumor cells were implanted into immunodeficient mice by left ventricular injection; as described in previous work, metastatic cells were extracted from the harvested brain metastases Source literature (Glutathione peroxidase 4-dependent glutathione high-consumption drives acquired platinum chemoresistance in lung cancer-derived brain metastasis). Human embryonic lung fibroblasts (HFL1) were from Hai Xing Bio, and human cerebral vascular pericytes were from Zhong Qiao Xin Zhou.

[0119] Culture of conventional cells: Conventional cells were cultured under the conditions of 37°C and 5% CO2, and were routinely adherently cultured using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin or the special medium for HFL1. When the confluence reached 75%, the cells were passaged or harvested.

[0120] Culture of lung cancer organoids: The main collection criteria are patients with clinically locally advanced or metastatic lung cancer, aged 18 or above, with fresh tissue obtained by biopsy or surgical resection of primary or metastatic lesions. The collected tumor tissue samples are used to generate tumor fragments about 1 cubic millimeter in size with sterilized ophthalmic scissors. They are suspended in cold Hank's balanced salt solution (HBSS) containing antibiotics and transported to the laboratory on ice within 1 hour after resection. After washing three times with cold HBSS containing antibiotics and slicing with a sterile blade, the samples are incubated in DMEM / F12 supplemented with 0.001% DNase, 1 mg / mL collagenase / dispase, penicillin-streptomycin, and 0.5 mg / mL amphotericin B at 37 °C for 2 hours, gently stirred and intermittently resuspended. Then, the digested tissue suspension is titrated by pipetting and passed through a 100-μm filter. Subsequently, the cell suspension generated from the tissue samples is centrifuged at 300 g for 3 minutes. Then, the pellet is resuspended in LCOM. Thereafter, 200 μL of Matrigel is added to 100 μL of the cell suspension to establish organoids, and the resulting suspension is inverted and solidified in a pre-warmed 6-well culture plate (Corning) at 37 °C for 30 minutes. After gelation, 3 mL of LCOM is added to each well. The medium is changed every two to three days.

[0121] Passage: The organoids are resuspended in TrypLE Express at 200 μL per well and incubated at 37 °C for 10 minutes to dissociate. Then, 10 mL of DMEM / F12 containing 10% FBS is added, and centrifuged at 300 × g for 5 minutes. The pellet is washed with DPBS and centrifuged at 112 × g for 3 minutes. 200 μL of Matrigel is added to 100 μL of the cell suspension to establish organoids, and re-seeded at a ratio of 1:3–1:4 to form new organoids.

[0122] Single-cell line culture in the chip: After digesting the cells with trypsin, the cell density is adjusted to 1×10^6 cells / ml. The cells are placed in the printer, and after determining the coordinate points, printed 6 times. After confirming the cell morphology under the microscope, the chip is placed in an incubator at 37 °C for 45 - 60 minutes, 75 μL of liquid is added, and thereafter the intracellular liquid is changed every 3 days.

[0123] In-chip organoid culture: Digest the cells with TrypLE and incubate in the incubator for ten minutes. Then place them in a low-speed centrifuge at 300g for 5 minutes. After removing the supernatant, wash the precipitate with cold DMEM / F12, place it in a low-speed centrifuge at 300g for 5 minutes for centrifugation. Add TrypLE to the tube again and incubate in the incubator for ten minutes to digest the cells, then centrifuge at 300g for 5 minutes. Add PBS to resuspend the cell suspension, adjust the density to 8000 cells / ml, place it in the printer, print 12 times. After determining the cell morphology under the microscope, place the chip in an incubator at 37°C for 45 - 60 minutes, add 75 μl of liquid, and then change the intracellular liquid every 3 days.

[0124] In-chip multicellular culture: Digest the co-cultured cells as described above and resuspend them in PBS to an appropriate cell concentration. Then place them in the printer successively and use different print heads to print different types of cells. The total printing time should be less than 30 minutes. After all the cells are printed, determine the cell morphology under the microscope, place the chip in an incubator at 37°C for 45 - 60 minutes, add 75 μl of liquid, and then change the intracellular liquid every 3 days.

[0125] Cell viability assay: After cell culture is completed, remove the culture medium in the chip. Subsequently, introduce a solution containing Calcein-AM / PI dyes (1 μmol L-1 Calcein-AM, 1 μmol L-1 PI) into the chip and incubate for 15 minutes under minimal light conditions. After removing the dye solution, reintroduce the stem cell culture medium. Then place the chip under an inverted fluorescence microscope for fluorescence scanning. The cell viability calculation formula is Ng / (Ng+Nr)×100%, where Ng and Nr are the numbers of green fluorescent cells and red fluorescent cells respectively.

[0126] Histology and immunohistochemistry: Fix the organoids and their corresponding parental tumors in 4% paraformaldehyde, then perform agarose embedding, sectioning, dewaxing, dehydration, and hematoxylin and eosin staining. Use antibodies against thyroid transcription factor (TTF-1), cytokeratin 7 (CK7), and Napsin A for LCO for immunohistochemistry (IHC) and capture images using an upright microscope.

[0127] Immunofluorescence assay: Tumor spheres for immunofluorescence imaging were placed in a culture dish and fixed with 4% paraformaldehyde solution for 20 min. After rinsing with PBS, the samples for nuclear protein staining were treated with 0.5% Triton X-100 solution for 10 min and then rinsed with PBS three times. Subsequently, the tumor spheres were blocked with 4% BSA solution for 30 min. Then the tumor spheres were incubated with the primary antibody overnight at 4 °C and, if necessary, with the fluorescently labeled secondary antibody for 2 h at room temperature. Finally, the tumor spheres were stained with DAPI for 10 min. Fluorescence imaging was performed after washing with PBS. The dilution ratios of the primary antibodies thyroid transcription factor (TTF-1) (Wuhan Sanying) was 1:200, α-SMA (Wuhan Sanying) was 1:800, CK7 (Zhengneng Biotech) was 1:100, and Collagen III (MCE) was 1:100. The secondary antibody used for detecting CK7 was CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (Wuhan Sanying), and the secondary antibodies used for detecting TTF-1, α-SMA, and Collagen III were CoraLite594–conjugated Goat Anti-Rabbit IgG(H+L) (Wuhan Sanying). The dilution ratio of all secondary antibodies was 1:100. The fluorescence intensity of the cells was measured using ImageJ software (https: / / imagej.net / ).

[0128] Live cell tracing: The cells were centrifuged and the supernatant was discarded (or cell tracers CFSE - green or C34565 - red were used). Then, the cells were suspended in the culture medium at a dilution ratio of 1:500 (or a 1:1000 dilution protocol with PBS could also be used), incubated in an incubator at 37 °C for 5 min, and then cooled in a refrigerator at 4 °C for 15 min. Subsequently, centrifugation was performed again (1000 RPM, 5 min) to remove the tracer in the supernatant, and then the cells were resuspended in the culture medium.

[0129] The experimental results (dynamic changes in cell viability on the chip) are as follows:

[0130] Experiment 1: Comparison of 3D printing and traditional inoculation of lung cancer organoids

[0131] Compared with the cell clusters generated by an inkjet printer, when using a pipette, it is difficult to ensure the aggregation of cells at the center point of the droplet, regardless of how small the volume is. This highlights the irreplaceability of inkjet printing technology in the accuracy of cell arrangement and operation. In the case of using a 3D printer, the printing of organoids can maximize the maintenance of cell morphology consistent with that in Matrigel. Through this technology, we can precisely control the arrangement of lung cancer organoids in a three-dimensional structure and achieve their precise spatial arrangement. Compared with traditional Matrigel culture, using a 3D printer can more accurately reproduce the microenvironment of cells in vivo, thus better simulating cell growth and interaction under physiological conditions. The breakthrough of this technology lies in that it can not only ensure the consistency of cell morphology but also achieve precise spatial arrangement, providing a more reliable and controllable experimental platform for organoid research. Specifically as Figure 7 shown.

[0132] Experiment 2: Isolation, culture and histological verification of lung cancer organoids

[0133] Patient-derived lung cancer organoids, samples for generating organoids were collected from 35 advanced cancer patients from October 24, 2023 to April 23, 2024. We successfully established 23 LCOs (lung cancer organoids) with a success rate of 65%, including 3 pleural effusion samples and 20 surgical tissue-derived samples.

[0134] Subsequently, these samples were embedded in agarose paraffin and then subjected to hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) to verify the organoids and compare their morphology and pathology with the original tissue. The IHC markers were selected from the markers commonly used for diagnosing lung cancer subtypes. H&E staining and IHC showed that the LCOs retained the pathological characteristics of the original tumor tissue. In addition, the LCOs derived from ADC (lung adenocarcinoma) expressed classical ADC markers, including thyroid transcription factor 1 (TTF-1) and napsin A. These data indicate that the LCOs can maintain the morphology and pathological characteristics of the original tumor and reflect its individual characteristics. Specifically as Figure 8 shown.

[0135] Experiment 3: Verification of cell viability of 3D-printed lung cancer organoids and microenvironmental stromal cells

[0136] To simulate the cellular tissue environment in real living organisms, especially for studying the metastatic microenvironment of tumors, understanding the long-term survival of cells in the model is crucial for simulating physiological conditions and disease development. Moreover, the long-term survival of cells in the model directly affects the stability and reliability of the model. If the survival rate of cells is lower than a certain level within seven days, it will affect the practicality and reproducibility of the model and reduce the research value of the model. This experiment uses a relatively long time span of seven days, during which changes in biological characteristics such as cell growth, differentiation, and interaction can be observed. By evaluating the survival of cells within seven days, a comprehensive understanding of the dynamic change process of cells in the model can be obtained, providing important reference data for subsequent research. Specifically, as Figure 9 Figure 2 shows a schematic diagram of the cell viability verification of 3D-printed lung cancer organoids and microenvironmental stromal cells: The cell viability of lung cancer organoids prepared using 3D printing technology was verified with different types of microenvironmental stromal cells. The cell viability was detected using an ampi viability kit at regular intervals every day, and relevant photos were recorded and taken.

[0137] Experiment 4: Three-dimensional growth of lung cancer and its microenvironment in a 3D printed mold and expression of marker proteins

[0138] Under the scanning of a confocal microscope of the chip after cell immunofluorescence staining, we observed that tumor cells and stromal cells showed the characteristics of 3D growth in the chip. It is worth noting that the TTF-1 and CK7 marker proteins of tumor cells and organoids, as well as aSMA of fibroblasts, were all shown to surround the DAPI-stained cell nuclei. Further observation showed that the positional relationship between the membrane protein CK7 and the nuclear protein TTF-1 was such that the green CK7 surrounded the outer layer of the red TTF-1, while the nuclear staining was blue, i.e., DAPI staining. This result strongly implies the complex three-dimensional structure of cells in the chip and provides strong support for subsequent research on cell interactions.

[0139] Compared with traditional culture methods, the growth of such cells in a 3D environment has important technical advantages and broad application prospects. 3D culture conditions are closer to the in vivo physiological environment, can better simulate tissue structure and cell-cell interactions, and provide a more accurate platform for the establishment of disease models and drug screening. In addition, through 3D culture, biological processes such as cell migration, invasion, and differentiation can also be studied, bringing new breakthroughs and development opportunities for biomedical research and clinical applications. Specifically, as Figure 10 shown.

[0140] Experiment 5: Spatial distribution of personalized lung cancer and its microenvironment using 3D bioprinting

[0141] In addition, 3D printing technology has not only achieved remarkable results in single-cell printing but also realized precise regulation of the distance between cells by using live cell tracers to characterize different types of cells and then printing them separately. Whether they are mixed, adjacent, or far apart, we can easily control the arrangement of cells, thus achieving precise cell-cell interactions when constructing models. This not only helps to deeply understand biological mechanisms but also provides more reliable research tools and platforms for fields such as disease research, drug screening, and tissue engineering. Therefore, achieving precise regulation of cell distance is of great significance and value in the application of 3D printing technology. As Figure 11 Shown is a schematic diagram of the spatial distribution of personalized lung cancer and the microenvironment using 3D bioprinting: By using 3D printing technology, two combinations of HFL1 and BrM3, and HFL1 and pericytes are printed separately, achieving their distribution patterns of being far apart (A), adjacent (B), and fused (C) in space.

[0142] Experiment 6: Visualization of the fusion and interaction between lung cancer organoids and the tumor microenvironment

[0143] Finally, we observed that when tumor cells and fibroblasts were printed simultaneously, the fibroblasts were gradually surrounded by the tumor cells, forming a special structure with tumor cells on the outer layer and fibroblasts on the inner layer. This special cell behavior cannot be observed in traditional 2D culture. Further, after replacing the tumor cells with organoids and performing cell immunofluorescence staining, a similar cell behavior was also observed. The importance of this discovery lies in providing us with a new perspective on cell-cell interactions and tissue structure formation. By observing the behavior of cells in 3D printed models, we can more deeply understand the interactions, signal transduction, and dynamic changes in tissue structure between tumor cells and normal cells. This helps to reveal the mechanisms of tumor occurrence and development, providing a deeper theoretical basis and practical guidance for tumor treatment and drug development. Therefore, the discovery of this function is of great significance for promoting tumor research and drug development. As Figure 12 Shown is a schematic diagram of observing the fusion and interaction between lung cancer organoids and the tumor microenvironment under an inverted fluorescence microscope using cell tracers: With the help of 3D printing technology, we achieved the adjacent distribution of HFL1 (fibroblasts) and H1299 (tumor cells). Over time, we observed that the fibroblasts were gradually surrounded by the tumor cells, forming a special structure with tumor cells on the outer layer and fibroblasts on the inner layer.

[0144] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible architectures, functions, and operations of systems, methods, and computer program products according to various embodiments of the present disclosure. In this regard, each block in the flowchart or block diagram may represent a module, a program segment, or a portion of code that contains one or more executable instructions for implementing a specified logical function. It should also be noted that in some alternative implementations, the functions marked in the blocks may occur in a different order than that marked in the accompanying drawings. For example, two consecutive blocks shown may actually be executed substantially in parallel, and they may sometimes be executed in the reverse order, depending on the functions involved. It should also be noted that each block in the block diagram and / or flowchart, as well as combinations of blocks in the block diagram and / or flowchart, may be implemented by a dedicated hardware-based system that performs the specified functions or operations, or may be implemented by a combination of dedicated hardware and computer instructions.

[0145] In general, the various example embodiments of the present disclosure may be implemented in hardware or a dedicated circuit, software, firmware, logic, or any combination thereof. Some aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that can be executed by a controller, a microprocessor, or other computing devices. When aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented as non-limiting examples in hardware, software, firmware, a dedicated circuit or logic, general hardware or a controller or other computing devices, or some combination thereof.

[0146] Those skilled in the art can clearly understand that for the convenience and brevity of description, the specific working processes of the systems, devices, and units described above can refer to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0147] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices, and methods may be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the units is only a logical function division, and there may be other division methods in actual implementation. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Another point is that the couplings or direct couplings or communication connections shown or discussed with each other may be indirect couplings or communication connections through some interfaces, devices, or units, and may be electrical, mechanical, or other forms.

[0148] The unit described as a separation component may or may not be physically separated. The component shown as a unit may or may not be a physical unit, that is, it may be located in one place or distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0149] In addition, each functional unit in various embodiments of the present invention may be integrated in a processing unit, or each unit may exist physically alone, or two or more units may be integrated in one unit. The above integrated unit may be implemented in the form of hardware or in the form of a software functional unit.

[0150] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. Those skilled in the art should understand that various modifications and combinations can be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A method for printing a microenvironment containing an organoid based on 3D printing, characterized in that: The method comprises: S1: Obtain the organoid cell suspension and microenvironment cell suspension to be printed; S2: placing the first suspension in a nozzle of a 3D printing device, determining a first printing position of the first suspension on the substrate through a microscope in the printing device, spraying the first suspension onto a PDMS chip in the printing device, and printing to obtain organoids or microenvironment cells; the first suspension is any one of an organoid and a microenvironment cell suspension; S3: placing the second suspension in the nozzle of the 3D printing device, determining the second printing position of the second suspension on the substrate through the microscope in the printing device, spraying the second suspension onto the PDMS chip in the printing device, and printing to obtain a microenvironment containing organoids; the second suspension is any one of the microenvironment and the organoid cell suspension, and the first suspension and the second suspension are different.

2. The method for printing a microenvironment containing an organoid based on 3D printing according to claim 1, characterized in that: When determining the second printing position in S3, determining the distance between the second printing position and the first printing position according to the development stage of the tumor cells, and determining the second printing position based on the first printing position and the distance; Alternatively, when determining the second printing position in S3, the distance between the second printing position and the first printing position is determined according to the research purpose, and the second printing position is determined based on the first printing position and the distance; Optionally, the first printing position and the second printing position are arranged at coordinate points at the micrometer or millimeter level; Optionally, the bottom layer of the PDMS chip is paved with a matrix substrate; Optionally, the distance between the nozzle and the host substrate is less than 0.3 cm.

3. The method for printing a microenvironment containing an organoid based on 3D printing according to claim 1, characterized in that: The method also includes: real-time monitoring of the state of the organoid invasion of the microenvironment cells in the organoid cell suspension and the microenvironment cell suspension by live cell fluorescence tracing technology under a microscope, and adjusting the components and conditions of the cell culture medium according to the state.

4. The method for printing a microenvironment containing an organoid based on 3D printing according to claim 1, characterized in that: After S3, the method further includes S4: cross-linking the microenvironment cells containing the organoids on the PDMS chip, and placing them in a cell culture medium for culture to obtain a 3D cell precipitation, which is a research model; Optionally, the cross-linking method includes: placing the PDMS chip in a dish and cross-linking at a first temperature for a first time period; Optionally, the diameter of the dish is 50 cm; Optionally, the first temperature is 37° C.; optionally, the first time period is 45 minutes.

5. The method for printing a microenvironment containing an organoid based on 3D printing according to claim 1, characterized in that: The method for preparing the organoid cell suspension comprises: S101-1: obtaining a tumor organoid sample; S101-2: washing the organoid sample with PBS to remove the surface serum, adding TrypLE to pipette and beat evenly, placing the sample in an incubator for digestion and neutralizing it with a neutralizing solution, and centrifuging the neutralized solution; S101-3: Add TrypLE to the neutralization solution precipitate after centrifugation in S101-2, put it into an incubator for digestion, and neutralize it with neutralization solution. The neutralization solution after centrifugation is the organoid cell suspension after resuspending; optionally, the organoid density in the resuspended liquid is greater than or equal to 4X10^4 cells / ml.

6. The method for printing a microenvironment containing an organoid based on 3D printing according to claim 1, characterized in that: The method for preparing the microenvironment cell suspension includes: S102-1: washing the microenvironment cells with PBS; S102-2: submerging the microenvironment cells with trypsin, placing them in an incubator for digestion, neutralizing them with a culture medium corresponding to the microenvironment cells and centrifuging them; S102-3: resuspending the precipitate after centrifugation in S101-2 to obtain the microenvironment cell suspension; optionally, the microenvironment cells include: H1299, HFL1.

7. The method for printing a microenvironment containing an organoid based on 3D printing according to claim 1, characterized in that: The PDMS chip is provided with a flow channel and a plurality of nozzles connected to the flow channel, and the cell suspension flows into the nozzles through the flow channel; the chip is moved along a preset path to sequentially spray the cell suspension in the plurality of nozzle groups to a designated position on the surface of the cell culture device to form a printed cell cluster, and one nozzle group includes one or more nozzles.

8. A computer device, characterized in that: The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 1 to 7.

9. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 1 to 7 are implemented.

10. A computer program product, comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the method described in any one of claims 1 to 7 are implemented.

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