A method for spatiotemporal 3D printing of tumor microenvironment organoids

Abstract

The application provides a method for printing a tumor microenvironment organoid in space-time 3D, comprising: printing a shell gel microsphere, and then printing an inner core gel microsphere in the shell gel microsphere to obtain a core-shell gel microsphere; performing irreversible cross-linking and solidification on the core-shell gel microsphere to form a stable structure, thereby obtaining a tumor microenvironment organoid model; and in the process of printing the inner core gel microsphere in the shell gel microsphere, performing time-dimension and / or space-dimension regulation printing to achieve the effect of constructing different tumor microenvironment characteristics. By printing the core-shell gel microsphere structure in space-time regulation, the organoid and stromal cells can be positioned in the inner core and the shell gel layer, respectively, so that the organoid model with different tumor microenvironment characteristics in a spatially layered architecture can be constructed, and the tumor cells can be guided to self-assemble, thereby achieving the effect of regulating the initial state of the tumor cells, and the tumor organoid can maintain a stable three-dimensional structure in the subsequent culture process.

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering technology, and in particular to a method for spatiotemporal 3D printing of tumor microenvironment organoids. Background Art

[0002] Patient-derived tumor organoids can highly reproduce the spatial morphology and similar pathophysiological characteristics of the source tumor tissue, and have become the most promising in vitro model for cancer disease research, drug screening and personalized medicine at this stage. The conventional method of culturing tumor organoids in vitro is to isolate tumor cells from patient-derived tumor tissue, use matrix gel to embed them to provide an extracellular matrix environment, and promote cell self-assembly to form a compact 3D tumor tissue structure. However, due to the uncontrollable self-assembly behavior of cells, the occurrence of tumor organoids in matrix gel is often random, and the number and morphology are different, with low reproducibility and consistency, which limits their clinical translation application. On the other hand, the progression of tumors is closely related to their microenvironment (stromal cells, immune cells, extracellular matrix and soluble factors, etc.). Among them, stromal cells (such as cancer-associated fibroblasts) account for a rich proportion, forming a physical wrapping layer for tumor cells, and regulating the drug response behavior of tumors. Therefore, the preparation of organoid models with tumor microenvironment characteristics is of great significance for cancer-related research.

[0003] Bio-3D printing technology can directional arrange cells, biomaterials, small molecules and other active substances, making it easy to quickly construct personalized in vitro three-dimensional biomimetic tissue structures, and has gradually become a core technology for the preparation of tumor organoids. Currently, methods for preparing tumor organoids based on bio-3D printing technology are limited by the following: (1) cells are randomly distributed in hydrogel materials, and the initial state of cells is uncontrollable, resulting in uneven scales of the formed organoids; (2) the constructed tumor microenvironment model usually prints a mixture of multi-component cells, lacking a tumor-stroma spatial hierarchical structure.

[0004] The above-mentioned record of background technology knowledge is intended to help ordinary technicians in this field understand the existing technology that is relatively close to the present invention, and at the same time facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clear that in the absence of clear evidence that the above-mentioned content has been disclosed before the filing date of this patent application, the above-mentioned background technology should not be used to evaluate the novelty of the technical solution of this application. Summary of the Invention

[0005] Technical issues

[0006] In order to solve the above problems, the purpose of the present invention is to provide a method for spatiotemporal 3D printing of tumor microenvironment organoids. By spatiotemporal modulation and printing of "core-shell" gel microbead structures, organoids and stromal cells can be positioned in the inner core and outer shell gel layers, respectively, to construct organoid models with different tumor microenvironment characteristics with spatial layered architectures. Tumor cell self-assembly can also be guided to achieve the effect of regulating the initial state of tumor cells. Tumor organoids can maintain a stable three-dimensional structure during subsequent culture.

[0007] The present invention includes the following aspects.

[0008] Scheme 1: A method for spatiotemporal 3D printing of tumor microenvironment organoids, comprising:

[0009] Shell gel microbeads are printed, and then inner core gel microbeads are printed inside the shell gel microbeads to obtain core-shell gel microbeads; the core-shell gel microbeads are irreversibly cross-linked and solidified to form a stable structure, thereby obtaining a tumor microenvironment organoid model.

[0010] Furthermore, during the process of printing the inner core gel microbeads inside the outer shell gel microbeads, the time dimension and / or space dimension are controlled and printed to achieve the effect of constructing different tumor microenvironment characteristics.

[0011] Furthermore, the time dimension regulated printing includes: after culturing the shell gel microbeads, selecting different time nodes during the culturing period to print the core gel microbeads.

[0012] Furthermore, the time dimension regulated printing includes: culturing the inner core gel microbeads after completing at least one printing, and selecting different time nodes during the culture period to print the inner core gel microbeads multiple times until the culture of the tumor microenvironment organoid model is completed.

[0013] Furthermore, the spatial dimension controlled printing includes: printing the core gel microbeads at different positions inside the shell gel microbeads by setting the printing coordinates and printing paths of the core gel microbeads.

[0014] Furthermore, before printing the inner core gel microbeads, the outer shell gel microbeads need to be three-dimensionally scanned and imaged and modeled into a three-dimensional outer shell microbead model, and then the three-dimensional outer shell microbead model is spatially divided to generate the printing coordinates and printing path of the inner core gel microbeads, and then the inner core gel microbeads are printed.

[0015] Furthermore, the tumor microenvironment organoid model that has completed at least one printing of inner core gel microbeads and cultured is subjected to three-dimensional scanning imaging and modeling into a three-dimensional organoid model. The three-dimensional organoid model is then spatially divided to generate the printing coordinates and printing path of the inner core gel microbeads, and then the inner core gel microbeads are printed.

[0016] Furthermore, the step of printing the shell gel microbeads includes: controlling the shell structure printing material to transform from a liquid state to a semi-gel state, utilizing the instantaneous contact between the extruded material and the receiving base plate during the printing process to form the shell gel microbeads, and at the same time setting the temperature of the receiving base plate to maintain the three-dimensional morphology of the shell gel microbeads.

[0017] Furthermore, the step of printing the shell gel microbeads specifically includes:

[0018] Step 2.1, loading the shell structure printing material into the extrusion printing nozzle, and controlling the temperature of the nozzle to transform the shell structure printing material from a liquid state to a semi-gel state;

[0019] Step 2.2: Set the distance between the print head and the receiving base plate to ensure that the bottom surface of the extruded shell structure printing material is in effective contact with the receiving base plate;

[0020] Step 2.3: Write G-code to control the movement of the print head and print related parameters;

[0021] Step 2.4: Utilize the instantaneous contact between the extruded material and the receiving base plate during the printing process to form the shell gel microbeads; and simultaneously set the temperature of the receiving base plate to maintain the three-dimensional morphology of the shell gel microbeads.

[0022] Furthermore, in step 2.1, the temperature of the nozzle is controlled in the range of 10-20°C.

[0023] Furthermore, in step 2.2, the distance between the print head and the receiving substrate is set in the range of 100-500 μm.

[0024] Furthermore, in step 2.2, the receiving bottom plate may be a culture well plate, which facilitates subsequent high-throughput drug screening.

[0025] Furthermore, in step 2.3, the printing-related parameters include air pressure, injection time, etc.

[0026] Furthermore, in step 2.4, the temperature of the receiving base plate is set in the range of 10-15°C.

[0027] Furthermore, the step of printing the inner core gel microbeads includes: controlling the inner shell structure printing material to transform from a liquid state to a semi-gel state, and at the same time setting the distance between the inner core structure material and the receiving base plate during extrusion to ensure that the printed inner core gel microbeads are embedded in the outer shell gel microbeads.

[0028] Furthermore, the step of printing the core gel microbeads specifically includes:

[0029] Step 2.5, performing three-dimensional scanning imaging and modeling of the shell gel microbeads into a three-dimensional shell microbead model;

[0030] Step 2.6: Divide the three-dimensional shell microbead model into spatial positions to generate the printing coordinates and printing path of the core gel microbeads;

[0031] Step 2.7: Load the core structure printing material onto another extrusion nozzle, control the temperature of the printing nozzle so that the core structure printing material changes from a liquid state to a semi-gel state; set the distance between the printing nozzle and the receiving base plate to ensure that the printed core gel microbeads are embedded in the shell gel microbeads; thereby completing the printing of the core-shell gel microbeads.

[0032] Furthermore, in step 2.5, the shell gel microbeads are subjected to three-dimensional scanning imaging and modeling, specifically by scanning the printed shell gel microbeads using a technology that can penetrate into the interior of the microspheres and observe the cell distribution, and obtaining a three-dimensional shell microbead model through modeling.

[0033] Furthermore, in step 2.7, the temperature of the print head is controlled in the range of 10-20°C.

[0034] Furthermore, in step 2.7, the distance between the printing nozzle and the receiving base plate is set to be 100-300 μm.

[0035] Combined with the spatiotemporally controlled printing of inner core gel microbeads, the "core-shell" gel microbeads can be cultured after completing one printing. During the culture period, different time nodes can be selected to print the inner core gel microbeads multiple times until the cultivation of the tumor microenvironment organoid model is completed. At this point, the spatiotemporally controlled printing and directional culture of the "core-shell" gel microbeads can be achieved.

[0036] Furthermore, the shell structure printing material is selected from at least one of methacrylated gelatin, gelatin, hyaluronic acid, and sodium alginate.

[0037] Furthermore, the core structure printing material is selected from at least one of matrix gel, gelatin, and collagen.

[0038] Furthermore, cells are added to the shell structure printing material.

[0039] Furthermore, cells are added to the core structure printing material.

[0040] Furthermore, the irreversible crosslinking curing method is selected from one of photocuring, ionic crosslinking or enzymatic crosslinking.

[0041] Furthermore, the culture medium involved in the culture process is prepared according to different tumor organoid types.

[0042] Furthermore, the spatiotemporal 3D printing method for tumor microenvironment organoids also includes: culturing core-shell gel microbeads, combining three-dimensional scanning imaging technology, and printing growth factors in specific areas of the organoids for targeted induction, so as to guide the proliferation trend and direction of the organoid cells.

[0043] Furthermore, the three-dimensional scanning imaging technology is a technology that can penetrate into the interior of the microspheres and observe the distribution of cells.

[0044] Furthermore, the three-dimensional scanning imaging technology includes OCT coherence tomography technology.

[0045] Furthermore, the spatiotemporal 3D printing method for tumor microenvironment organoids also includes: replacing the receiving base plate with a culture well plate, and adding different concentrations of drugs into each well of the culture well plate to perform high-throughput drug screening.

[0046] Solution 2: A 3D printing system comprising a printer, a print head, and a 3D scanning and imaging device, both mounted on the printer. The printer is configured to print; the print head is configured to deposit printing material for the outer shell structure and / or the inner core structure; and the 3D scanning and imaging device is configured to scan and image the printed gel microbeads. This 3D printing system implements at least one step of the aforementioned method for spatiotemporal 3D printing of tumor microenvironment organoids.

[0047] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0048] Beneficial effects

[0049] According to the present invention, a method for constructing a homogenized tumor microenvironment organoid model using a gel microbead embedded printing strategy is proposed. This method forms "core-shell" gel microbeads by printing biomaterials containing different cell types twice or more in combination. The constructed "core-shell" gel microbeads have the following advantages:

[0050] 1) By positioning organoids and stromal cells in the core and shell gel layers, respectively, a spatially hierarchical microenvironmental organoid model was constructed;

[0051] 2) Using the core hydrogel geometry to “set fixed boundaries” for tumor organoids, guiding tumor cell self-assembly and achieving the effect of regulating the initial state of tumor cells;

[0052] 3) The mechanically strong shell hydrogel provides a stable three-dimensional support environment, allowing tumor organoids to maintain a stable three-dimensional structure during subsequent culture.

[0053] 4) By combining OCT coherence tomography technology to perform three-dimensional imaging, modeling, and regional division of the shell gel microbeads or core-shell gel microbeads, the printing nozzle can locate and print specific areas, and the printed core gel microbeads or cytokines can be regulated in the dimensions of time and space to construct organoid models with different tumor microenvironment characteristics.

[0054] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the existing technology and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to make the above-mentioned and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the following is a brief introduction to the drawings required for use in the specific embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0056] Figure 1 Schematic diagram showing the longitudinal section of the three-dimensional shell microbead model and the generated spatial coordinates;

[0057] Figure 2 A diagram showing the experimental results of gel microbeads with a "core-shell" structure constructed based on spatiotemporal 3D printing;

[0058] Figure 3 A diagram showing the experimental results of preparing tumor cell clusters based on "core-shell" structured gel microbeads;

[0059] Figure 4 A statistical diagram showing the scale of tumor cell clusters prepared based on "core-shell" structured gel microbeads;

[0060] Figure 5 Schematic diagram and experimental results of the tumor microenvironment organoid model constructed based on the one-time molding of "core-shell" gel microbeads and the time-space molding of "core-shell" gel microbeads. DETAILED DESCRIPTION

[0061] Those skilled in the art may refer to the contents herein and appropriately substitute and / or modify the process parameters to achieve the desired effect. However, it should be noted that all such substitutions and / or modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products and preparation methods described herein have been described through preferred embodiments. It is obvious that those skilled in the art can modify or appropriately change and combine the products and preparation methods described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0062] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention utilizes the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will control.

[0063] Unless otherwise specified, the materials, methods, and examples described herein are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0064] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other unless there is a conflict.

[0065] It should be understood that any technical solution claimed for protection in the present invention does not involve the diagnosis and treatment of diseases.

[0066] In addition, the experimental methods used in the examples are conventional methods unless otherwise specified. The materials and reagents used are commercially available unless otherwise specified. Reagents or instruments used without manufacturer indication are commercially available conventional products. All publications and other references cited herein are incorporated by reference in their entirety.

[0067] The present invention is described in detail below.

[0068] Example 1:

[0069] A preparation process of core-shell gel microbeads is provided, the specific steps comprising:

[0070] Step 1: Preparation of shell hydrogel material and core hydrogel material.

[0071] Among them, the shell hydrogel material is preferably methacrylated gelatin (GelMA) with a concentration of 8%, containing phenyl-2.4.6-trimethylbenzoyl lithium phosphite (LAP) with a concentration of 0.4% as a photoinitiator. During the preparation, an appropriate amount of LAP powder is weighed according to the specific material concentration, and dissolved in cell culture medium (conventional culture medium) as a solvent. The appropriate methacrylated gelatin solid is added to the dissolved LAP solution. After sufficient dissolution, the corresponding shell hydrogel printing precursor solution is obtained. As needed, matrix cells (fibroblasts) are resuspended in the precursor solution to prepare the shell structure printing material; the core hydrogel material is preferably collagen with a concentration of 10 mg / mL. During the preparation process, the collagen mother solution is diluted with gelatin solution (concentration of 10%) to prepare the required core structure printing material, and cells are added as needed. The specific process is to weigh an appropriate amount of gelatin powder and dissolve it with cell culture medium to obtain a gelatin solution. According to needs, the gelatin solution is mixed with the collagen mother liquor in different volumes to obtain a composite hydrogel material. Tumor organoid seed cells are resuspended based on the composite material to obtain the required core structure printing material.

[0072] Step 2: Construct gel microbeads with a "core-shell" structure, namely core-shell gel microbeads, by printing microbead combinations twice.

[0073] To facilitate observation, a yellow dye was added to the shell structure printing material, and a red dye was added to the core structure printing material. Two micro-extrusion nozzles were installed in the shell and core structure printing materials, respectively, with a 6-well culture plate as the base. The shell printing nozzle temperature was set at 18°C, causing the shell structure printing material to transition from a liquid to a semi-gel state. The nozzle was set at a distance of 200μm from the plate, with a given pneumatic pressure of 100kPa and an extrusion time of 500ms. Given a constant nozzle diameter and shell printing material concentration, the size of the shell gel microbeads could be controlled by controlling the shell structure printing material extrusion time.

[0074] The shell gel microbeads were scanned and modeled by optical coherence tomography (OCT technology) to obtain a three-dimensional shell microbead model, and its spatial position was divided, such as Figure 1 , generate the printing coordinates and printing path of the core gel microbeads.

[0075] Switch to the print head loaded with the core structure printing material, control the temperature of the print head at 20℃, change the core structure printing material from liquid to semi-gel state, set the distance between the print head and the receiving base plate at 300μm, execute the printing path of the generated core gel microbeads, and ensure that the core gel microbeads are printed inside the shell microbeads. The results are as follows Figure 2 shown.

[0076] When printing the inner core gel microbeads, combined with OCT scanning imaging, the inner core gel microbeads can be regulated and printed in the time or space dimension. That is, for regulation in the time dimension, the outer shell gel microbeads can be cultured, and different time nodes can be selected during the culture period. The three-dimensional outer shell microbead model can be obtained by combining OCT scanning imaging and modeling, and then the inner core gel microbeads can be printed; for regulation in the space dimension, the three-dimensional outer shell microbead model can be obtained by combining OCT scanning imaging and modeling, and its printing coordinates and spatial position can be divided, and then the inner core gel microbeads can be printed; if regulation in the time and space dimensions are performed simultaneously, the three-dimensional model can be obtained by combining OCT scanning imaging and modeling, and the core-shell gel microbeads can be printed multiple times.

[0077] Alternatively, during the culture process, the core-shell gel microbeads can be combined with OCT scanning technology to print growth factors to specific regions of the organoid during culture, thereby localizing and inducing them to guide the trend and direction of organoid cell proliferation. For high-throughput drug screening, varying concentrations of drugs can be added to each well of the culture plate containing the printed core-shell gel microbeads.

[0078] After the core-shell gel microbeads were printed, a 405 nm wavelength blue light source (power 50 mW / cm 2 ) for 30 s to achieve irreversible curing of the core-shell gel microbeads through photocrosslinking.

[0079] Subsequently, the core-shell gel microbeads were incubated in an incubator for 2 hours to complete the solidification of the inner core collagen material and the liquefaction of the gelatin material, and then culture medium was added for subsequent culture.

[0080] Example 2:

[0081] A method for spatiotemporal 3D printing of tumor microenvironment organoids is provided, specifically, a method for bio-3D printing of "core-shell" gel microbeads to construct a uniformly sized tumor organoid model. The printing steps are the same as in Example 1, wherein the shell structure printing material is 8% GelMA containing 0.5% LAP photoinitiator. The core printing material is a collagen / gelatin solution loaded with human pancreatic cancer cells, with a cell density of 1×10 7 cells / mL. First, the shell bio-ink was printed to form gel microbeads, and then the coordinates were located using OCT technology to complete the printing of the core microbeads. At the same time, the core bio-ink containing human pancreatic cancer cells was directly printed to form a single gel bead as a control. Figure 3As shown in the figure, the tumor cells in the "core-shell" gel microbeads were restrained by the inner core microbead structure and showed a convergent state on the third day of culture. After 5 days of culture, a tumor cell cluster was formed. In the single gel beads, the tumor cells were randomly distributed. After 3 days of culture, the proliferation of the dispersed tumor cells was not obvious. After 5 days of culture, multiple tumor cell clusters of uneven sizes were formed. The size statistics of the tumor cell clusters cultured with "core-shell" gel microbeads were performed, and the results are shown in the figure. Figure 4 As shown, the average diameter of the cultured tumor cell clusters was 100.4 μm, with a small standard deviation (about 5.9), indicating that the tumor cell clusters prepared based on this method had good size uniformity.

[0082] Example 3:

[0083] Provide spatiotemporal controlled printing to construct tumor organoid models with different stromal microenvironments.

[0084] The specific printing steps are the same as those in Example 1, wherein the shell structure printing material is 8% GelMA containing 0.5% LAP photoinitiator, and loaded with human fibroblasts with a cell density of 4×10 6 cells / mL, and the core printing material is a collagen / gelatin solution containing tumor cells from tumor patients.

[0085] The printing process is as follows: first, two groups of fibroblast-loaded shell microbeads are printed. One group is immediately printed with the inner core tumor bio-ink, and the other group is cultured for 2 days before printing the inner core tumor bio-ink.

[0086] The experimental results are as follows Figure 5 As shown, the fibroblasts in the uncultured shell microbeads are discrete spheres (serial number 1), while the fibroblasts in the cultured shell microbeads gradually extend to form spindles (serial number 2). After 4 days of culture, the two co-culture models formed tumor organoid models with different matrix microenvironment characteristics.

[0087] Example 4:

[0088] Tumor microenvironment organoid models constructed by printing gel microbeads using different methods are used for drug testing. Studies have reported that the uneven size of tumor cell clusters in hydrogels can affect the accuracy of drug testing results. Previous studies used single-bead hydrogels to culture tumor organoids. Since the positional distribution of tumor cells in the hydrogel is random, this results in the formation of multiple tumor cell clusters of uneven sizes, which limits the application of drug testing. However, by using the "core-shell" gel microbeads of the present invention to culture tumor organoids, the geometric structure of the inner core hydrogel "sets fixed boundaries" for the tumor organoids, guiding the self-assembly of tumor cells to achieve the effect of regulating the initial state of tumor cells. Tumor cell clusters of uniform size can be prepared, which helps to improve the accuracy of drug testing. In addition, a large number of studies have shown that the stromal microenvironment can enhance the drug resistance of tumor cells. Therefore, the introduction of the stromal microenvironment will more accurately reflect the drug response behavior of tumors. In this example, gel microbeads were printed using different methods to construct a single-bead pancreatic cancer monoculture model, a core-shell microbead pancreatic cancer monoculture model, and a core-shell microbead stromal microenvironment tumor model (i.e., a co-culture model of pancreatic cancer cells and fibroblasts). After one week of culture, the cells were treated with 50µM gemcitabine for 48 hours. After the drug treatment, cell survival rates in the three models were measured to assess the drug response of the tumors. The cell survival rates and standard deviations between the models are shown in Table 1.

[0089] Table 1 - Drug testing results of tumor microenvironment organoid models constructed by printing gel microbeads using different methods

[0090]

[0091] As can be seen from Table 1, the cell survival rates detected in the three samples of the single-bead cultured tumor model have large differences, while the single-culture model and co-culture model cultured with core-shell microbeads have smaller standard deviations; at the same time, for the same drug concentration, the co-culture model shows a larger cell survival rate, indicating that the matrix microenvironment promotes the drug resistance of tumor cells.

[0092] Example 5:

[0093] A 3D printing system includes a printer, a print head, and a 3D scanning and imaging device, both mounted on the printer. The printer is configured to print; the print head is configured to deposit printing material for the outer shell structure and / or the inner core structure; and the 3D scanning and imaging device is configured to scan and image the printed gel microbeads. The 3D printing system implements at least one step of the aforementioned method for spatiotemporal 3D printing of tumor microenvironment organoids and achieves the same technical effects. To avoid repetition, this embodiment will not be described in detail.

[0094] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0095] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope defined by the appended claims.

[0096] Although the present invention has been described in detail and certain specific embodiments have been cited, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

[0097] Although the above-mentioned specific embodiments have shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, replacements and changes can be made to the form and details of the described devices or methods without departing from the spirit of the present disclosure. In addition, the various features and methods described above can be used independently of each other, or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above-mentioned embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and their obvious modifications and equivalents. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0098] Matters not covered in the present invention are all known technologies.

Claims

1. A method for spatiotemporal 3D printing of tumor microenvironment organoids, characterized in that include: Printing shell gel microbeads, and then printing core gel microbeads inside the shell gel microbeads, performing controlled printing in the time dimension and / or space dimension to obtain core-shell gel microbeads; Irreversibly cross-linking and curing the core-shell gel microbeads to form a stable structure, thereby obtaining a tumor microenvironment organoid model; The time dimension controlled printing includes: after culturing the shell gel microbeads, selecting different time points during the culturing period to print the core gel microbeads; Before printing the core gel microbeads, the shell gel microbeads need to be three-dimensionally scanned and imaged and modeled into a three-dimensional shell microbead model, and then the three-dimensional shell microbead model is spatially divided to generate the printing coordinates and printing path of the core gel microbeads, and then the core gel microbeads are printed; or Performing three-dimensional scanning and imaging of the tumor microenvironment organoid model that has completed at least one printing of core gel microbeads and cultured, and modeling it into a three-dimensional organoid model, then spatially dividing the three-dimensional organoid model, generating the printing coordinates and printing path of the core gel microbeads, and then printing the core gel microbeads; The step of printing the shell gel microbeads comprises: controlling the shell structure printing material to change from a liquid state to a semi-gel state, utilizing the instantaneous contact between the extruded material and the receiving base plate during the printing process to form the shell gel microbeads, and simultaneously setting the temperature of the receiving base plate to maintain the three-dimensional morphology of the shell gel microbeads; and / or The step of printing the core gel microbeads includes: controlling the inner shell structure printing material to change from a liquid state to a semi-gel state, and setting the distance between the core structure material and the receiving base plate during extrusion to ensure that the printed core gel microbeads are embedded in the outer shell gel microbeads; The shell structure printing material is selected from at least one of methacrylated gelatin, gelatin, hyaluronic acid, and sodium alginate; the core structure printing material is selected from at least one of matrix gel, gelatin, and collagen; and cells are added to the shell structure printing material and / or the core structure printing material.

2. The method according to claim 1, wherein: During the process of printing the core gel microbeads inside the shell gel microbeads, the printing is regulated in the time dimension and / or space dimension to achieve the effect of constructing different tumor microenvironment characteristics.

3. The method according to claim 2, wherein: After completing at least one printing of inner core gel microbeads, the microbeads are cultured, and multiple printings of inner core gel microbeads are performed at different time points during the culture period until the culture of the tumor microenvironment organoid model is completed.

4. The method according to claim 2 or 3, characterized in that: The spatial dimension controlled printing includes: setting the printing coordinates and printing paths of the inner core gel microbeads so that the inner core gel microbeads are printed at different positions inside the outer shell gel microbeads.

5. The method according to claim 1, wherein: It also includes culturing core-shell gel microbeads, combining them with three-dimensional scanning imaging technology to print growth factors in specific areas of organoids for targeted induction, so as to guide the proliferation trend and direction of organoid cells.

6. The method according to claim 1, wherein: The method also includes replacing the receiving bottom plate with a culture well plate, and adding drugs of different concentrations into each well of the culture well plate to perform high-throughput drug screening.

7. A 3D printing system comprising a printer, a print head, and a 3D scanning imaging device, wherein the print head and the 3D scanning imaging device are both mounted on the printer; The printer is used to perform printing operations; the print head is used to print the outer shell structure material and / or the inner core structure material; the three-dimensional scanning imaging device is used to scan and image the printed gel microbeads; it is characterized in that: the 3D printing system implements the method of spatiotemporal 3D printing of tumor microenvironment organoids according to any one of claims 1-6.

Images