Method for space-time 3D printing of tumor microenvironment organoid

Abstract

The invention provides a space-time 3D printing method for tumor microenvironment organoid, which comprises the following steps: printing shell gel microbeads, and printing core gel microbeads in the shell gel microbeads to obtain core-shell gel microbeads; carrying out irreversible cross-linking curing on the core-shell gel microbeads to form a stable structure, so as to obtain the tumor microenvironment organ-like model. In the process of printing the inner core gel microbeads in the shell gel microbeads, time dimension and / or space dimension regulation and control printing is carried out, so that the effect of constructing different tumor microenvironment characteristics is achieved. The'core-shell 'gel microbead structure is printed through space-time modulation, organoids and stromal cells can be positioned in the inner core gel layer and the outer shell gel layer respectively, organoid models of different tumor microenvironment characteristics of a space layered structure are constructed, self-assembly of tumor cells can be guided, the effect of regulating the initial state of the tumor cells is achieved, and the tumor microenvironment characteristic of the tumor cells is improved. The tumor organoid can keep 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, provide an extracellular matrix environment with the help of matrix gel embedding, and promote cell self-assembly to form a compact 3D tumor-like 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 its 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 which stromal cells (such as cancer-associated fibroblasts) account for a large proportion, forming a physical encapsulation layer for tumor cells, regulating the drug response behavior of tumors. Therefore, the preparation of an organoid model with tumor microenvironment characteristics is of great significance for cancer-related research.

[0003] Bio-3D printing technology can orient active substances such as cells, biomaterials, and small molecules, and is easy to quickly construct personalized in vitro three-dimensional biomimetic tissue structures, gradually becoming the core technology for the preparation of tumor organoids. At present, the methods for preparing tumor organoids based on bio-3D printing technology are limited by: (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 multi-component cells together, lacking a tumor-stroma spatial hierarchical architecture.

[0004] The above-mentioned record of background technical knowledge is intended to help ordinary technicians in this field understand the existing technologies that are 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 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 printing the "core-shell" gel microbead structure through spatiotemporal regulation, the organoids and stromal cells can be positioned in the inner core and outer shell gel layers respectively, and an organoid model with different tumor microenvironment characteristics of a spatially layered architecture can be constructed. The self-assembly of tumor cells can 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.

[0006] The present invention includes the following aspects.

[0007] Scheme 1, a method for spatiotemporal 3D printing of tumor microenvironment organoids, comprising: The shell gel microbeads are printed, and then the 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.

[0008] Furthermore, in 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.

[0009] Furthermore, the time dimension regulated printing includes: after culturing the shell gel microbeads, selecting different time nodes during the culturing period to print the inner core gel microbeads.

[0010] Furthermore, the time dimension regulated printing includes: culturing the inner core gel microbeads after completing at least one inner core gel microbead 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.

[0011] Furthermore, the spatial dimension controlled printing includes: printing the inner core gel microbeads at different positions inside the outer shell gel microbeads by setting the printing coordinates and printing paths of the inner core gel microbeads.

[0012] 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.

[0013] Furthermore, the tumor microenvironment organoid model that has completed at least one inner core gel microbead printing and culture is three-dimensionally scanned and imaged and modeled into a three-dimensional organoid model, and then the three-dimensional organoid 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.

[0014] Furthermore, the step of printing the shell gel microbeads includes: 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 setting the temperature of the receiving base plate to maintain the three-dimensional morphology of the shell gel microbeads.

[0015] Furthermore, the step of printing the shell gel microbeads specifically includes: 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; 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; Step 2.3, write G-code to control the movement of the print head and print related parameters; Step 2.4, utilizing the instantaneous contact between the extruded material and the receiving base plate during the printing process to form 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.

[0016] Furthermore, in step 2.1, the temperature of the nozzle is controlled in the range of 10-20°C.

[0017] Furthermore, in step 2.2, the distance between the print head and the receiving substrate is set in the range of 100-500 μm.

[0018] Furthermore, in step 2.2, the receiving bottom plate may be a culture well plate, which facilitates subsequent high-throughput drug screening.

[0019] Furthermore, in step 2.3, the printing-related parameters include air pressure, injection time, etc.

[0020] Furthermore, in step 2.4, the temperature of the receiving bottom plate is set in the range of 10-15°C.

[0021] Furthermore, the step of printing the inner 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 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.

[0022] Furthermore, the step of printing the inner core gel microbeads specifically includes: Step 2.5, performing three-dimensional scanning imaging and modeling of the shell gel microbeads into a three-dimensional shell microbead model; Step 2.6, dividing the spatial position of the three-dimensional shell microbead model to generate the printing coordinates and printing path of the core gel microbead; Step 2.7, load the core structure printing material on another extrusion nozzle, control the temperature of the printing nozzle so that the core structure printing material changes from liquid to 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.

[0023] 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.

[0024] Furthermore, in step 2.7, the temperature of the print head is controlled in the range of 10-20°C.

[0025] Furthermore, in step 2.7, the distance between the print head and the receiving base plate is set to be 100-300 μm.

[0026] Combined with the spatiotemporal controlled printing of inner core gel microbeads, after completing one printing of the "core-shell" gel microbeads, they can be cultured. During the culture period, different time nodes can be selected to print the inner core gel microbeads multiple times until the culture of the tumor microenvironment organoid model is completed. At this point, the spatiotemporal controlled printing and directional culture of the "core-shell" gel microbeads can be achieved.

[0027] Furthermore, the shell structure printing material is selected from at least one of methacrylated gelatin, gelatin, hyaluronic acid, and sodium alginate.

[0028] Furthermore, the inner core structure printing material is selected from at least one of matrix gel, gelatin and collagen.

[0029] Furthermore, cells are added to the shell structure printing material.

[0030] Furthermore, cells are added to the core structure printing material.

[0031] Furthermore, the irreversible crosslinking curing method is selected from photocuring, ionic crosslinking or enzymatic crosslinking.

[0032] Furthermore, the culture medium involved in the culture process is prepared according to different tumor organoid types.

[0033] Furthermore, the method of spatiotemporal 3D printing of tumor microenvironment organoids also includes: culturing the core-shell gel microbeads, combining three-dimensional scanning imaging technology, and printing growth factors in specific areas of the organoids for localized induction, so as to guide the proliferation trend and direction of the organoid cells.

[0034] 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.

[0035] Furthermore, the three-dimensional scanning imaging technology includes OCT coherence tomography technology.

[0036] Furthermore, the spatiotemporal 3D printing method for tumor microenvironment organoids also includes: replacing the receiving base 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.

[0037] Solution 2, a 3D printing system, the 3D printing system includes a printer, a print head, and a 3D scanning imaging device, both of which are installed on the printer; wherein the printer is used to perform a printing operation; the print head is used to print the outer shell structure printing material and / or the inner core structure printing material; the 3D scanning imaging device is used to scan and image the printed gel microbeads. The 3D printing system implements at least one step of the aforementioned method of spatiotemporal 3D printing of tumor microenvironment organoids.

[0038] Based on the common knowledge in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0039] Beneficial Effects According to the present invention, a method for constructing a homogenized tumor microenvironment organoid model by embedding gel microbeads in printing is proposed, which is to form "core-shell" gel microbeads by printing biomaterials carrying different types of cells twice or more in combination. The constructed "core-shell" gel microbeads have the following advantages: 1) A spatially hierarchical microenvironment organoid model was constructed by positioning organoids and stromal cells in the inner core and outer shell gel layers, respectively; 2) Using the inner core hydrogel geometry to “set fixed boundaries” for tumor organoids, guide tumor cell self-assembly, and achieve the effect of regulating the initial state of tumor cells; 3) Using the shell hydrogel with a certain mechanical strength to provide a stable three-dimensional support environment, the tumor organoids can maintain a stable three-dimensional structure during the subsequent culture process; 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 is used to locate and print specific areas, and the printed core gel microbeads or cytokines are regulated in the dimensions of time and space to construct organoid models with different tumor microenvironment characteristics.

[0040] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the prior art and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to make the above and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the drawings required for use in the specific implementation of the present invention will be briefly introduced below. 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 paying creative work.

[0042] Figure 1 A schematic diagram showing the longitudinal section of the three-dimensional shell microbead model and the generated spatial coordinates; Figure 2 A diagram showing the experimental results of gel microbeads with a "core-shell" structure constructed based on spatiotemporal 3D printing; Figure 3 A diagram showing the experimental results of preparing tumor cell clusters based on "core-shell" structured gel microbeads; Figure 4 A statistical diagram showing the scale of tumor cell clusters prepared based on “core-shell” structured gel microbeads; Figure 5 Schematic diagram of the effects 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

[0043] Those skilled in the art can refer to the content of this article and appropriately replace and / or modify the process parameters to achieve the same. However, it should be particularly noted that all similar replacements 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 in the present invention have been described through preferred examples. It is obvious that relevant personnel can modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] 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 the invention belongs. The present invention uses 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, shall prevail.

[0045] Unless specifically stated, 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 still described herein.

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without 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 without conflict.

[0047] It should be understood that any technical solution claimed for protection in the present invention does not involve the diagnosis and treatment of diseases.

[0048] In addition, the test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially. All the publications and other references mentioned in this article are incorporated herein by reference in their entirety.

[0049] The present invention is described in detail below.

[0050] Embodiment 1: A preparation process of core-shell gel microbeads is provided, and the specific steps include: Step 1: Preparation of shell hydrogel material and core hydrogel material.

[0051] Among them, the shell hydrogel material preferably has a concentration of 8% methacryloyl gelatin (GelMA), containing 0.4% phenyl-2.4.6-trimethylbenzoyl lithium phosphite (LAP) as a photoinitiator. In the preparation, an appropriate amount of LAP powder is weighed according to the specific material concentration, and the cell culture medium (conventional culture medium) is used as a solvent to dissolve it. The appropriate methacryloyl gelatin solid is added to the dissolved LAP solution. After sufficient dissolution, the corresponding shell hydrogel printing precursor solution is obtained. The matrix cells (fibroblasts) are resuspended in the precursor solution as needed to prepare the shell structure printing material; the core hydrogel material preferably has a concentration of 10 mg / mL collagen. In the preparation process, the collagen mother solution is diluted with a 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 in cell culture medium to obtain a gelatin solution, mix the gelatin solution with the collagen mother solution in different volumes as needed to obtain a composite hydrogel material, and resuspend tumor organoid seed cells based on the composite material to obtain the required core structure printing material.

[0052] Step 2: Construct gel microbeads with a "core-shell" structure, namely core-shell gel microbeads, by printing microbead combinations twice.

[0053] In order to facilitate observation, yellow dye is added to the shell structure printing material, and red dye is added to the core structure printing material. Two micro-extrusion nozzles are installed on the shell and core structure printing materials respectively, and a 6-well culture plate is used as the bottom plate. The shell printing nozzle temperature is 18°C, so that the shell structure printing material changes from a liquid state to a semi-gel state. The print nozzle is set at a distance of 200μm from the plate, the pneumatic pressure is given to be 100kPa, and the extrusion time is 500ms. Under the condition of a certain print nozzle diameter and shell printing material concentration, the size of the shell gel microbeads can be controlled by controlling the shell structure printing material extrusion time.

[0054] The shell gel microbeads are scanned and modeled by optical coherence tomography (OCT technology) to obtain a three-dimensional shell microbead model, and its spatial position is divided, such as Figure 1 , generate the printing coordinates and printing path of the core gel microbeads.

[0055] Switch to another print head loaded with 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.

[0056] 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, different time nodes can be selected during the culture period, and a 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, OCT scanning imaging and modeling can be combined to obtain a three-dimensional outer shell microbead model, and its printing coordinates and spatial position can be divided, and then the inner core gel microbeads can be printed; if both the time and space dimensions are regulated at the same time, a three-dimensional model can be obtained by combining OCT scanning imaging and modeling, and the core-shell gel microbeads can be printed multiple times.

[0057] Alternatively, during the culture process of the core-shell gel microbeads, the OCT scanning imaging technology can be combined as needed to print growth factors in specific areas of the organoids during the culture process to induce localization, so as to guide the proliferation trend and direction of the organoid cells. If high-throughput drug screening is to be performed, different concentrations of drugs can be added to each well of the culture plate printed with the core-shell gel microbeads.

[0058] 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.

[0059] 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.

[0060] Embodiment 2: A method for spatiotemporal 3D printing of tumor microenvironment organoids is provided, specifically, a bio-3D printing "core-shell" gel microbead to construct a tumor organoid model with uniform scale. The printing steps are the same as those in Example 1, wherein the outer shell structure printing material is 8% GelMA containing 0.5% LAP photoinitiator. The inner 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 by OCT technology and the core microbeads were printed; 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 convergence state on the third day of culture, and formed a tumor cell cluster after 5 days of culture; while in the single gel beads, the tumor cells were randomly distributed, and the proliferation of the dispersed tumor cells was not obvious after 3 days of culture, and multiple tumor cell clusters of uneven sizes were formed after 5 days of culture. The size statistics of the tumor cell clusters cultured with the "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 have good size uniformity.

[0061] Embodiment 3: Provide spatiotemporal controlled printing to construct tumor organoid models with different matrix microenvironments.

[0062] 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 inner core printing material is a collagen / gelatin solution containing tumor cells from tumor patients.

[0063] The printing process is to first complete the printing of two groups of fibroblast-loaded shell microbeads, one of which is immediately printed with inner core tumor bio-ink, and the other group is cultured for 2 days before printing with inner core tumor bio-ink.

[0064] 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.

[0065] Embodiment 4: The tumor microenvironment organoid model constructed by printing gel microbeads using different methods is used for drug testing. Studies have reported that the uneven size of tumor cell clusters in hydrogels will affect the accuracy of drug testing results. Previous studies used single-bead hydrogels to culture tumor organoids. Due to the random distribution of tumor cells in the hydrogel, multiple tumor cell clusters of uneven sizes were formed, which limited the application of drug testing. However, the "core-shell" gel microbeads of the present invention are used to culture tumor organoids. The inner core hydrogel geometric structure is used to "set fixed boundaries" for tumor organoids, guide tumor cell self-assembly, and 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 matrix microenvironment can enhance the drug resistance of tumor cells, so the introduction of the matrix microenvironment will more accurately reflect the drug response behavior of tumors. In this embodiment, gel microbeads were printed by different methods to construct a pancreatic cancer monoculture tumor model cultured by single beads, a pancreatic cancer monoculture tumor model cultured by core-shell microbeads, and a matrix microenvironment tumor model cultured by core-shell microbeads (i.e., a co-culture model of pancreatic cancer tumor cells and fibroblasts). After one week of culture, 50 μM gemcitabine was used for 48 hours. After the drug effect ended, the cell survival rate in the three models was detected to evaluate the drug response of the tumor. The results of cell survival rate and standard deviation between models are shown in Table 1.

[0066] Table 1 - Drug testing results of tumor microenvironment organoid models constructed by printing gel microbeads using different methods

[0067] As can be seen from Table 1, the cell survival rates detected in the three samples of the tumor model cultured with single beads have great 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.

[0068] Embodiment 5: A 3D printing system, the 3D printing system includes a printer, a print head, and a 3D scanning imaging device, wherein the print head and the 3D scanning imaging device are installed on the printer; wherein the printer is used to perform a printing operation; the print head is used to print a shell structure printing material and / or a core structure printing material; and the 3D scanning imaging device is used to scan and image the printed gel microbeads. The 3D printing system implements at least one step of the aforementioned method of spatiotemporal 3D printing of tumor microenvironment organoids, and can achieve the same technical effect. To avoid repetition, this embodiment will not be repeated.

[0069] 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.

[0070] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0071] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0072] Although the above-mentioned specific embodiments have been shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes can be made to the form and details of the described device or method without departing from the spirit of the present disclosure. In addition, the above-mentioned various features and methods 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 obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0073] 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 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.

2. The method according to claim 1, characterized in that: In 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.

3. The method according to claim 2, characterized in that: The control printing of the time dimension includes: After the outer shell gel microbeads are cultured, the inner core gel microbeads are printed at different time points during the culture period; or After completing at least one printing of inner core gel microbeads, the inner core gel 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 3, characterized in that: 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; or The tumor microenvironment organoid model that has completed at least one inner core gel microbead printing and culture is three-dimensionally scanned and imaged and modeled 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.

6. The method according to claim 1, characterized in that: 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 bottom plate during the printing process to form the shell gel microbeads, and setting the temperature of the receiving bottom plate to maintain the three-dimensional morphology of the shell gel microbeads; and / or The step of printing the inner core gel microbeads comprises: controlling the inner shell structure printing material to change from a liquid state to a semi-gel state, and setting the distance between the inner core structure material and the receiving bottom plate when it is extruded to ensure that the printed inner core gel microbeads are embedded in the outer shell gel microbeads.

7. The method according to claim 6, characterized in that: The shell structure printing material is selected from at least one of methacrylated gelatin, gelatin, hyaluronic acid, and sodium alginate; and / or The core structure printing material is selected from at least one of matrix gel, gelatin and collagen; and / or Cells are added to the shell structure printing material and / or the core structure printing material.

8. The method according to claim 1, characterized in that: It also includes the cultivation of core-shell gel microbeads, combined 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.

9. The method according to claim 1, characterized in that: 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.

10. 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 installed on the printer; wherein: 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 at least one step of the method for spatiotemporal 3D printing of tumor microenvironment organoids according to any one of claims 1-9.

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