Hydrogels, bioinks, organoids constructed therewith, drug screening models, and methods of construction

Abstract

The application relates to the technical field of biotechnology or biomedical engineering, in particular to a hydrogel, a biological ink, an organoid constructed by the same, a drug screening model and a construction method. The hydrogel comprises 3-4 wt% of gelatin, 0.5-2 wt% of sodium alginate and 20-40 wt% of Matrigel and a cell culture medium. The hydrogel helps to improve the bionics degree of a tumor microenvironment, can be used to prepare a biological ink containing target cells such as tumor cells, in particular can be used to prepare a biological ink of primary cells (such as primary renal cell carcinoma cancer cells), and an organoid model constructed by the biological ink under suitable 3D biological printing conditions, the organoid model is uniform in growth, fast in cell growth speed, high in consistency with parent tissues, can reduce the amplification time of patient-derived tumor cells in vitro and reduce the influence of subculture factors on the cell parent nature, helps to accelerate the drug development process and reduce the production cost.

Description

Technical Field

[0001] This application relates to the fields of biotechnology or biomedical engineering technology, and in particular to a hydrogel, bio-ink, organoids constructed therefrom, drug screening models, and construction methods thereof. Background Technology

[0002] Isolated cells typically lack the ability to maintain genomic integrity. Their inherent genomic instability makes them highly susceptible to gene alterations and damage during proliferation, leading to genetic variation and genomic evolution, which can interfere with the effectiveness of drug testing. Therefore, research on the construction of patient-derived tumor models requires not only a thorough understanding of the genotype and phenotypic characteristics of the original tumor tissue, but also the ability to maintain them stably, accurately, and reliably in in vitro models. Furthermore, exploring methods for constructing highly biomimetic tumor microenvironments that fully replicate the parental information in patients, from cellular composition to tissue structure, and achieving personalized, heterogeneous tumor model manufacturing based on patient-derived tumor cells to conduct relevant chemotherapy / targeted drug screening and evaluation, are also scientific challenges in the field of biomanufacturing. In view of this, this application is hereby submitted. Summary of the Invention

[0003] Based on this, one or more embodiments of this application provide a hydrogel, a bio-ink, organoids constructed therefrom, a drug screening model, a construction method, and applications. The technical solutions include the following:

[0004] One or more embodiments of this application provide a hydrogel comprising 3 wt%-4 wt% gelatin, 0.5 wt%-2 wt% sodium alginate and 20 wt%-40 wt% matrix gel, and cell culture medium.

[0005] In some embodiments of this application, the cell culture medium includes organoid culture medium.

[0006] One or more embodiments of this application also provide a bio-ink, the bio-ink comprising:

[0007] (1) the hydrogel, and,

[0008] (2) Target cells, wherein the cell culture medium is suitable for culturing the target cells.

[0009] In some embodiments of this application, the bio-ink satisfies one or more of the following conditions:

[0010] 1) The target cells include tumor cells; optionally, the tumor cells include renal cell carcinoma cells; optionally, the tumor cells include primary cells; and,

[0011] 2) The density of the target cells in the bio-ink is 1×10⁻⁶. 5 Cells / mL - 1×10 7 per mL.

[0012] One or more embodiments of this application provide a bioprinting product, the bioprinting product comprising:

[0013] (I) the hydrogel or the bio-ink described above; and...

[0014] (II) Other hydrogels or other bio-inks, among which,

[0015] The other hydrogels include 4.5 wt% to 5.5 wt% of methacrylamide gelatin, an initiator, and cell culture medium as defined above, and the other bioinks include the other hydrogels and target cells as defined above.

[0016] In some embodiments of this application, the bioprinted article satisfies one or more of the following conditions:

[0017] I) The initiator includes a photoinitiator; optionally, the photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphonite;

[0018] II) The initiator is used in the other bio-ink at an amount of 0.1 wt%-0.2 wt%; and,

[0019] III) The density of the target cells in the bio-ink is 1×10⁻⁶. 5 Cells / mL - 1×10 7 per mL.

[0020] One or more embodiments of this application provide a method for preparing an organoid model, wherein the preparation method uses the hydrogel or the bio-ink described above.

[0021] In some embodiments of this application, the preparation method includes the following steps: using the bio-ink for 3D bioprinting, culturing, and preparing organoid models.

[0022] In some embodiments of this application, the organoid model has a three-dimensional mesh structure with a size of (2-20)×(2-20)×(1-6) mm. 3 The number of layers ranges from 2 to 20, and the average diameter of the gel filaments is 400μm-500μm.

[0023] In some embodiments of this application, the step of 3D bioprinting the organoid model satisfies one or more of the following conditions:

[0024] (A) Use a 25G dispensing needle;

[0025] (B) The bio-ink is kept at 12.5℃-13.5℃ for 4-6 minutes before printing;

[0026] (C) Stack the code layer by layer according to the instructions in the computer-programmed G-code file;

[0027] (D) The fill mode is grid fill;

[0028] (E) The filler density is 20%-50%;

[0029] (F) Printing speed is 15mm / s-30mm / s;

[0030] (G) Extrusion pressure is 3-6 Bar;

[0031] (H) Extrusion speed is 2 mm / s-5 mm / s; and,

[0032] (I) The pullback distance is 0.5mm-1.2mm.

[0033] In some embodiments of this application, the cultivation satisfies one or more of the following conditions:

[0034] A) The temperature is 36℃-38℃;

[0035] B) Conducted in a carbon dioxide atmosphere;

[0036] C) The culture medium includes cell culture medium as defined above; and,

[0037] D) The culture medium should be changed every 2-3 days for 12-16 days.

[0038] One or more embodiments of this application provide an organoid model prepared by the preparation method described above.

[0039] One or more embodiments of this application provide a method for constructing a drug screening model, the method comprising:

[0040] Organoid models were prepared using the aforementioned preparation method; and,

[0041] Target cells from the organoid model were collected, 3D bioprinted, solidified, and used to prepare a drug screening model.

[0042] In some embodiments of this application, the 3D bioprinted drug screening model satisfies one or more of the following conditions:

[0043] (a) The bio-ink used is another bio-ink as defined above;

[0044] (b) The bio-ink used should be kept at 12.5℃-13.5℃ for 4-6 minutes before printing;

[0045] (c) Print according to the computer-programmed G-code file;

[0046] (d) Use a 27G dispensing needle;

[0047] (e) The drug screening model is droplet-shaped; optionally, the maximum surface diameter is 4mm-5mm.

[0048] (f) The receiving container includes a cell culture plate; optionally, the receiving surface of the receiving container is covered with a hydrogel, the hydrogel comprising 2 wt%-3 wt% methacryloyl gelatin and cell culture medium as defined above;

[0049] (g) The extrusion speed is 0.5 mm / s-1.5 mm / s; and,

[0050] (h) The printing pause time between holes is 2s-4s, and the retraction time is 0.5s-1.5s.

[0051] In some embodiments of this application, the step of collecting target cells in the organoid model includes:

[0052] The organoid model was washed, lysed, and cell clusters were collected; and,

[0053] The cell clusters were digested using a cell digestion solution to prepare a cell suspension.

[0054] In some embodiments of this application, the step of collecting target cells from the organoid model satisfies one or more of the following conditions:

[0055] a) The cleaning solution used includes PBS buffer;

[0056] b) The lysis conditions include: the lysis buffer used includes sodium chloride lysis buffer, 50mM-60mM sodium citrate and 15mM-25mM EDTA, and the time is 4-6 minutes.

[0057] c) The cell clusters are collected by centrifugation; optionally, the centrifugation conditions include a rotation speed of 1400 rpm-1600 rpm and a time of 4-6 minutes; and,

[0058] d) The digestion conditions include: the cell digestion solution contains 0.2 mg / mL to 0.4 mg / mL collagenase IV, the temperature is 35℃ to 38℃, and the time is 5 minutes to 10 minutes.

[0059] One or more embodiments of this application provide a drug screening model constructed using the described construction method.

[0060] One or more embodiments of this application provide a method for detecting a drug, the method comprising the steps of bringing the drug to be tested into contact with the drug screening model, and the step of detecting the activity of the target cells in the drug screening model after contact.

[0061] In some embodiments of this application, the detection method satisfies one or more of the following conditions:

[0062] (i) The drug to be tested includes one or more of tanciolimus, cabozantinib, everolimus, cisplatin and epirubicin;

[0063] (ii) The contact time is 40-55 hours;

[0064] (iii) The concentration of the test drug in contact with the sample is 0 μM-5000 μM; and,

[0065] (iv) Activity was detected using the CCK8 kit.

[0066] Compared to traditional technologies, the beneficial effects of this application include:

[0067] This application provides a formulated hydrogel that enhances the biomimicry of the tumor microenvironment. This hydrogel can be used to prepare bio-inks containing target cells, such as tumor cells, and particularly for preparing bio-inks containing primary cells (e.g., primary renal cell carcinoma cells). Organoid models constructed using this bio-ink under suitable 3D bioprinting conditions exhibit uniform growth, rapid cell growth, and high consistency with parental tissues. This reduces the in vitro expansion time of patient-derived tumor cells and mitigates the impact of passage culture factors on cell parentage, thus accelerating drug development and reducing production costs. Furthermore, this organoid model can provide a cell source for subsequent high-throughput 3D bioprinted drug screening models. Based on this, the required amount of clinical samples can be reduced in the construction of personalized drug screening models for clinical oncology patients. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0069] Figure 1Flowchart for the invention and design of personalized renal cell carcinoma organoid models for bioprinting;

[0070] Figure 2 Rheological property analysis of gelatin / sodium alginate / matrix-based bio-ink; including: A. Curves of G′ and G″ as a function of temperature under different material concentration ratios; B. Curves of G′ and G″ as a function of time under different material concentration ratios; C. Curves of G′ and G″ as a function of temperature for composite hydrogel material with optimal material ratio and pure matrix adhesive;

[0071] Figure 3 Comparison of cell growth states under three different culture conditions. Scale bar, 100 μm.

[0072] Figure 4 Comparison of HE and Ki67 staining of parental tumor tissue samples from 5 patients and bioprinted renal cell carcinoma organoid samples; scale bar, 20 μm;

[0073] Figure 5 Immunofluorescence staining images of E-cad, CD44 and CD133 dry markers in different BP-Organoids; scale bar, 20 μm;

[0074] Figure 6 Immunofluorescence staining images of E-cad and CD44 in different fields of view in BP-Organoids 1; scale bar, 20 μm;

[0075] Figure 7 To perform whole-exome sequencing analysis on renal cell carcinoma tissue samples and corresponding printed organoid models from 5 patients; (A) a graph showing the proportion of common / tumor-specific tumor-related mutations between printed organoid samples and tumor tissue samples; (B) a heatmap showing the correlation of SNV mutations between printed organoid samples and tumor tissue samples; (C) a graph showing the synonymous / non-synonymous mutation characteristics of renal cell carcinoma-related driver genes in printed organoid samples and tumor tissue samples; (D) a graph showing the proportion of different exon mutation types between printed organoid samples and tumor tissue samples.

[0076] Figure 8 Transcriptome sequencing analysis results for tumor tissue and organoid models;

[0077] Figure 9 This is an integrated image of a high-content, live / dead cell stained pore structure using laser confocal microscopy. Green represents live cells, and red represents dead cells.

[0078] Figure 10 The efficacy response curves of different chemotherapy / targeted drugs are shown for different patient samples. Detailed Implementation

[0079] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0081] the term

[0082] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0083] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0084] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0085] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0086] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0087] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0088] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0089] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0090] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0091] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0092] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0093] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0094] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0095] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0096] Cancer is a genetic disease, resulting from polygenic disorders caused by mutations in one or more genes within a cell. A key characteristic is the genomic heterogeneity and instability within the tumor. This genomic heterogeneity ultimately leads to heterogeneity in phenotypes, clinicopathological features, disease progression, and treatment outcomes in cancer patients, which can be categorized into intratumoral heterogeneity and intertumoral heterogeneity.

[0097] Traditional organoid culture techniques using gel embedding can effectively maintain the heterogeneity of patient-derived tumor organoids and provide screening capabilities that directly match clinical drug outcomes, making them feasible for widespread clinical application. However, there are currently few reports on mature technologies for culturing renal cell carcinoma organoids (renal cell carcinoma organoid models) that maintain parental genetic characteristics and applying them to drug detection. Furthermore, the limitations of traditional organoid culture methods, such as the difficulty in creating complex tissue structures with biomimetic multicellular components and the need for manual manipulation, pose challenges in quality control and scalability. This results in issues such as functional immaturity, low throughput, and significant batch-to-batch variability, requiring the integration of other technologies to meet the requirements of high-throughput practical applications.

[0098] This application primarily utilizes 3D bioprinting technology to explore novel alternatives to the manual construction of renal cell carcinoma organoids. It enhances the organoid construction technology in terms of automation and biomimicry, and combines the pathological subtype omics characteristics of clinical patients with next-generation sequencing technology to assess the heterogeneity and parental integrity of the models. Simultaneously, it performs batch screening of chemotherapy / targeted drugs based on high-throughput 3D bioprinted drug screening models. A retrospective analysis of the drug screening results and the phenotypic and genotypic results of the printed renal cell carcinoma organoids is conducted to elucidate the heterogeneous response mechanisms of different drugs, thus advancing the treatment from a universal (one-size-fits-all) approach to personalized treatment.

[0099] The beneficial effects of the embodiments of this application include: This application provides a hydrogel with optimized formulation, which helps to improve the biomimicry of the tumor microenvironment and can be used to prepare bio-inks containing target cells such as tumor cells, especially bio-inks for primary cells (such as primary renal cell carcinoma cells). Organoid models constructed with this bio-ink under suitable 3D bioprinting conditions grow uniformly, have a fast cell growth rate, and high consistency with parental tissues. This can reduce the expansion time of patient-derived tumor cells in vitro and reduce the impact of passage culture factors on cell parentage, which helps to accelerate the drug development process and reduce production costs. In addition, this organoid model can provide cell sources for subsequent high-throughput 3D bioprinted drug screening models. Based on this, the demand for clinical samples can be reduced in the construction of personalized drug screening models for clinical tumor patients. The beneficial effects of the embodiments of this application also include: (1) Combining two novel technologies, 3D bioprinting and organoids, to construct a new generation of methods for constructing personalized tumor models in vitro. (2) Replacing traditional animal experiments with personalized renal cell carcinoma 3D bioprinted models to avoid species specificity and shorten the experimental cycle.

[0100] First aspect of the embodiments of this applicationA hydrogel is provided, the hydrogel comprising 3 wt%-4 wt% gelatin, 0.5 wt%-2 wt% sodium alginate and 20 wt%-40 wt% matrix gel, and cell culture medium.

[0101] In the hydrogel of this application, the amount of gelatin used is, for example, 3 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, and 4 wt%, the amount of sodium alginate used is, for example, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, and 2 wt%, and the amount of matrix gel used is, for example, 22 wt%, 24 wt%, 26 wt%, 28 wt%, 30 wt%, 32 wt%, 34 wt%, 36 wt%, 38 wt%, and 40 wt%.

[0102] This application does not impose any particular limitation on the type of cell culture medium. Appropriate cell culture medium can be selected according to the needs. In the case of using the hydrogel to prepare bio-ink for bioprinting, the cell culture medium of this application can be an organoid culture medium, including but not limited to the complete culture medium involved in the embodiments of this application.

[0103] Second aspect of the embodiments of this application A bio-ink is also provided, the bio-ink comprising:

[0104] (1) the hydrogel, and,

[0105] (2) Target cells, wherein the cell culture medium is suitable for culturing the target cells.

[0106] The hydrogel in this application is used in conjunction with the target cells. The cell culture medium in the hydrogel is suitable for the culture of the target cells, and the hydrogel with the corresponding culture medium can be matched according to the type of target cells.

[0107] This application does not specifically limit the type of target cells, including but not limited to tumor cells. In some examples of this application, the tumor cells include renal cell carcinoma cells; in some examples of this application, the tumor cells include primary cells.

[0108] This application does not impose a specific limitation on the density of target cells in the bio-ink, including but not limited to controlling the cell density to 1×10⁻⁶. 5 Cells / mL - 1×10 7 cells / mL, for example, 1×10 5 cells / mL, 2×10 5cells / mL, 4×10 5 cells / mL, 6×10 5 cells / mL, 8×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 4×10 6 cells / mL, 6×10 6 cells / mL, 8×10 6 cells / mL, 1×10 7 per mL.

[0109] A second aspect of the embodiments of this application, A bioprinting product is provided, the bioprinting product comprising:

[0110] (I) the hydrogel or the bio-ink described above; and...

[0111] (II) Other hydrogels or other bio-inks, among which,

[0112] The other hydrogels include 4.5 wt% to 5.5 wt% of methacrylamide gelatin, an initiator, and cell culture medium as defined above, and the other bioinks include the other hydrogels and target cells as defined above.

[0113] This application does not specifically limit the type of initiator, including but not limited to photoinitiators. In some examples, the photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphonite.

[0114] This application does not impose a particular limitation on the amount of initiator used. The amount of initiator can be determined in combination with the type of initiator selected. In some examples, the amount of the initiator used in the other bio-ink is 0.1wt%-0.2wt% (e.g., 0.1wt%, 0.12wt%, 0.14wt%, 0.16wt%, 0.18wt%, 0.2wt%).

[0115] This application does not specifically limit the density of the target cells in other bio-inks, including but not limited to controlling the cell density to 1×10⁻⁶. 5 Cells / mL - 1×10 7 cells / mL, for example, 1×10 5 cells / mL, 2×10 5 cells / mL, 4×10 5 cells / mL, 6×10 5 cells / mL, 8×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 4×10 6cells / mL, 6×10 6 cells / mL, 8×10 6 cells / mL, 1×10 7 per mL.

[0116] Third aspect of the embodiments of this application A method for preparing an organoid model is provided, wherein the preparation method uses the hydrogel or the bio-ink described above.

[0117] This application does not specifically limit the method for preparing organoid models. In some examples, the preparation method includes the following steps: 3D bioprinting using the bio-ink, culturing, and preparing organoid models.

[0118] The renal cell carcinoma model constructed using the method described in this application exhibits good growth, with tumor cells proliferating and growing rapidly and uniformly, and showing high consistency with the parental tumor tissue, including morphological consistency, intratumoral and intertumoral heterogeneity, and stable genetic information.

[0119] In some examples of this application, the organoid model has a three-dimensional mesh structure with a size of (2-20)×(2-20)×(1-6) mm. 3 The organoid model has 2 to 20 layers, with an average diameter of 400 μm to 500 μm for the gel filaments. In this application, the organoid model has a length of, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 mm; a width of, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 mm; a height of, for example, 1, 2, 3, 4, 5, or 6 mm; and a number of layers of, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The average diameter of the gel filaments is 400, 420, 440, 460, 480, or 500 μm.

[0120] In some examples of this application, the step of 3D bioprinting the organoid model satisfies one or more of the following conditions:

[0121] (A) Use a 25G dispensing needle;

[0122] (B) The bio-ink is kept at 12.5℃-13.5℃ (e.g., 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5℃) for 4-6 minutes (e.g., 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6 minutes) before printing; the purpose of keeping it warm is to make the extruded biofilm water in a gel-like linear state.

[0123] (C) Stack the code layer by layer according to the instructions in the computer-programmed G-code file;

[0124] (D) The fill mode is grid fill;

[0125] (E) Filler density is 20%-50% (e.g., 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%);

[0126] (F) Printing speed is 15mm / s-30mm / s (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30mm / s);

[0127] (G) Extrusion pressure is 3-6 bar (e.g., 3, 3.5, 4, 4.5, 5, 5.5, 6 bar);

[0128] (H) Extrusion speed is 2 mm / s-5 mm / s (e.g., 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 4, 4.2, 4.4, 4.6, 4.8, 5 mm / s); and,

[0129] (I) The pullback distance is 0.5mm-1.2mm (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2mm).

[0130] This application does not impose specific limitations on the cultivation conditions; appropriate cultivation conditions can be selected based on actual needs. In some examples of this application, the cultivation meets one or more of the following conditions:

[0131] A) Temperatures between 36℃ and 38℃ (e.g., 36, 36.2, 36.4, 36.8, 37, 37.2, 37.4, 37.6, 37.8, 38℃);

[0132] B) Conducted in a carbon dioxide atmosphere;

[0133] C) The culture medium includes cell culture medium as defined above; and,

[0134] D) The culture time is 12-16 days (e.g., 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16 days), and the culture medium is changed every 2-3 days.

[0135] Fourth aspect of the embodiments of this application An organoid model is provided, which is prepared by the preparation method described above.

[0136] Fifth aspect of the embodiments of this applicationA method for constructing a drug screening model is provided, the method comprising:

[0137] Organoid models were prepared using the aforementioned preparation method; and,

[0138] Target cells from the organoid model were collected, 3D bioprinted, solidified, and used to prepare a drug screening model.

[0139] In some examples of this application, the 3D bioprinted drug screening model satisfies one or more of the following conditions:

[0140] (a) The bio-ink used is another bio-ink as defined above;

[0141] (b) The bio-ink used is kept at 12.5°C-13.5°C (e.g., 12.5, 13, 13.5°C) for 4-6 minutes (e.g., 4, 4.5, 5, 5.5, 6 minutes) before printing;

[0142] (c) Print according to the computer-programmed G-code file;

[0143] (d) Use a 27G dispensing needle;

[0144] (e) The drug screening model is droplet-shaped; optionally, the maximum surface diameter is 4 mm-5 mm (e.g., 4, 4.2, 4.4, 4.6, 4.8, 5 mm);

[0145] (f) The receiving container includes a cell culture plate; optionally, the receiving surface of the receiving container is coated with a hydrogel, the hydrogel comprising 2 wt%-3 wt% (e.g., 2, 2.2, 2.4, 2.6, 2.8, 3 wt%) of methacryloyl gelatin and cell culture medium as defined above;

[0146] (g) Extrusion speed of 0.5 mm / s-1.5 mm / s (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 mm / s); and,

[0147] (h) The printing pause time between holes is 2 to 4 seconds (e.g., 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 seconds), and the retraction time is 0.5 to 1.5 seconds (0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 seconds).

[0148] In some examples of this application, the steps of collecting target cells in the organoid model include:

[0149] The organoid model was washed, lysed, and cell clusters were collected; and,

[0150] The cell clusters were digested using a cell digestion solution to prepare a cell suspension.

[0151] In some examples of this application, the step of collecting target cells in the organoid model satisfies one or more of the following conditions:

[0152] a) The cleaning solution used includes PBS buffer;

[0153] b) The lysis conditions include: using a lysis buffer consisting of sodium chloride lysis buffer and 50 mM-60 mM (e.g., 50, 52, 54, 56, 58, 60 mM) sodium citrate and 15 mM-25 mM (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM) EDTA for a time of 4 to 6 minutes (e.g., 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6 minutes);

[0154] c) The method of collecting the cell clusters includes centrifugation; optionally, the centrifugation conditions include a rotation speed of 1400 rpm to 1600 rpm (e.g., 1400, 1450, 1500, 1550, 1600 rpm) and a time of 4 minutes to 6 minutes (e.g., 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6 minutes); and,

[0155] d) The digestion conditions include: the cell digestion solution containing 0.2 mg / mL to 0.4 mg / mL (e.g., 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4 mg / mL), a temperature of 35°C to 38°C (e.g., 35, 35.5, 36, 36.5, 37, 37.5, 38°C), and a time of 5 minutes to 10 minutes (e.g., 5, 6, 7, 8, 9, 10 minutes).

[0156] Sixth aspect of the embodiments of this application A drug screening model is provided, which is constructed using the aforementioned construction method.

[0157] Seventh aspect of the embodiments of this application A method for detecting a drug is provided, the method comprising the steps of bringing the drug to be tested into contact with the drug screening model, and the step of detecting the activity of the target cells in the drug screening model after contact.

[0158] In some examples of this application, the detection method satisfies one or more of the following conditions:

[0159] (i) The drug to be tested includes one or more of tanciolimus, cabozantinib, everolimus, cisplatin and epirubicin;

[0160] (ii) The exposure time is 40-55 hours (e.g., 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55 hours);

[0161] (iii) The concentration of the test drug in contact with the sample is 0 μM-5000 μM (e.g., 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 μM); and,

[0162] (iv) Activity was detected using the CCK8 kit.

[0163] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0164] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0165] Addressing the needs of precision medicine, this application's embodiments utilize 3D bioprinting technology to construct highly biocompatible in vitro personalized tumor organoid models and drug screening models using patient-derived tumor cells and biomimetic matrix materials. These models are then used for evaluating the efficacy of anti-tumor drugs, aiming to provide new technical means and research platforms for personalized tumor treatment. The main components include:

[0166] (1) By selecting appropriate bio-inks and 3D bioprinting processes, the structure and function of the patient's tumor tissue can be reproduced in vitro, and a personalized organoid model with a biomimetic microenvironment and a parental similarity to the tumor in vivo can be obtained.

[0167] (2) The tissue morphology, exon and transcriptome sequencing omics analysis of personalized renal cell carcinoma organoid models were performed to evaluate the genetic stability of single nucleotide polymorphism mutations and insertion and deletion mutations. The expression of tumor driver genes was also compared to identify their parental nature and ability to maintain intratumoral and intertumoral heterogeneity.

[0168] (3) While ensuring the accuracy of the organoid model, tumor cells from the expanded and cultured renal cell carcinoma organoids were recovered and used for high-throughput 3D bioprinting of drug screening models. The drug screening models were then used for clinical first-line / second-line chemotherapy / targeted drug screening.

[0169] Combination Figure 1 The embodiments of this application will be explained and described below:

[0170] 1. Tissue samples and cells

[0171] In this embodiment, the five tissue samples from renal cell carcinoma patients are numbered 1 to 5, and the organoid model and drug screening model constructed subsequently are also numbered 1 to 5.

[0172] Each renal cell carcinoma tissue sample was divided into four aliquots: one aliquot for deoxyribonucleic acid (DNA) extraction for whole exome sequencing (WES), one aliquot for ribonucleic acid (RNA) extraction for transcriptome sequencing (RNA-seq), and one aliquot for direct immunohistochemical analysis. These three operations were used to record the biological and genetic information of the parental tumor tissue. One tissue sample was used to digest and extract tumor cells for 3D printing of a cross-shaped three-dimensional mesh structure and its amplification culture (i.e., operation 2 below). The steps for extracting tumor cells were referenced in the following literature:

[0173] Na JC,Kim J,Kim SY,et al.Establishment of patient-derived three-dimensional organoidculture in renal cell carcinoma[J].Investigative andClinical Urology,2020,61(2):216.

[0174] That is, the obtained renal cell carcinoma tissue samples are further mechanically processed with surgical scissors until they are reduced to 0.1-0.5 mm. 3 The tissue fragments were then collected, and cancer cells were extracted. The main steps included:

[0175] (1) Transfer the shredded tissue fragments to a 15mL centrifuge tube, add 10mL of ice-cold DMEM / F12 medium, gently pipette up and down with a 10mL pipette, then place on ice and let the tissue fragments settle naturally. Remove 7.5mL of supernatant.

[0176] (2) After repeating the above cleaning steps, centrifuge at 1500 rpm for 5 minutes, remove as much culture medium as possible, add 4-5 mL of preheated digestion solution per gram of tissue fragments, and digest in a 37°C, carbon dioxide incubator for 2-3 hours; wherein, the digestion solution is: collagenase IV (Type-IV collagenase) enzyme solution with a collagenase IV content of 0.3 mg / mL.

[0177] (3) After digestion, the volume of the digested product was increased to 15 mL with ice-cold DMEM / F12 medium, filtered through a 70 μm filter sieve, and then ice-cold DMEM / F12 medium was added to 50 mL.

[0178] (4) Centrifuge at 1500 rpm for 5 minutes, set the centrifuge temperature to 8℃, discard the supernatant, add ice-cold DMEM / F12 medium to 15 mL, and transfer to a 15 mL centrifuge tube;

[0179] (5) Centrifuge at 1500 rpm for 5 minutes, set the centrifuge temperature to 8℃, resuspend the cells in complete culture medium, and count the cells with a cell counter (Countstar) for the next experiment.

[0180] The complete culture medium used in this application contains: Advanced DMEM / F12 medium, 10 mM HEPES buffer, 1% glutamine, 1×B-27 serum-free additive, 1 mM N-acetylcysteine, 500 nM MA83-01 (transforming growth factor β-kinase type 1 receptor inhibitor), 20 μg / mL epidermal growth factor (EGF) + 10 μg / mL basic fibroblast last growth factor (bFGF), 10 μM Y-27632 (ROCK inhibitor), 1% P / S, and 0.1% bovine serum albumin (BSA).

[0181] 2. Printing organoid models (i.e., 3D mesh structures)

[0182] In the specific model printing process, the bio-ink used was a multi-component hydrogel material system containing complete culture medium, 3.75 wt% gelatin (Sigma; G1890), 1 wt% sodium alginate (Sigma; A0682), and 30 wt% matrix gel (BD; 354234). The tumor cell density was 3 × 10⁻⁶. 6 per mL.

[0183] Bio-ink was drawn into a 3mL syringe, fitted with a 25G (standard, 0.26mm inner diameter) dispensing needle, and loaded into the printing sleeve of a desktop extrusion 3D bioprinter (SUNP BIOMAKER). The syringe was incubated at 15℃ for 5 minutes to ensure the extruded bio-ink formed a straight gel. Printing was then performed under conditions where the bio-ink had been confirmed to form a smooth, straight gel through micro-extrusion. The printing path followed the computer-programmed G-code file instructions, layer by layer; infill mode: mesh infill; infill density: 30%; printing speed: 20mm / s; extrusion pressure: 5Bar; extrusion speed: 4mm / s; pullback distance: 0.6mm. The final printed hydrogel 3D mesh structure measured 10×10×2mm. 3 It consists of six layers, with an average diameter of 500 μm for the gel filaments.

[0184] 2.1 Optimization of Bio-ink

[0185] This application uses a gelatin / sodium alginate / matrix multi-element bio-ink for the printing and construction of personalized in vitro tumor models.

[0186] Regarding the selection of gelatin concentration, the commonly used gelatin concentrations in the literature are 5wt%, 7wt%, and 10wt%. Considering that the printed cells are sensitive primary cells, the over-gelling state during high-concentration gelatin printing will cause primary cell death. Therefore, a low concentration of 3.75wt% gelatin was selected to reduce the viscosity of the bio-ink and the shear stress felt by the cells during the printing process.

[0187] The printing concentration of sodium alginate is usually 1 wt%, but because it has a certain degree of biological inertness, it cannot provide a biological site for cell growth to adhere, resulting in cells being unable to attach and spread, and some adherent cells may even abort and undergo apoptosis.

[0188] This application adds a matrix gel component to gelatin and sodium alginate to create a more biomimetic tumor extracellular matrix microenvironment. Due to limited reports on mixed-printing of matrix gels, to clarify the optimal concentration of matrix gel, rheological properties were analyzed for multi-component hydrogel material systems containing 3.75 wt% gelatin, 1 wt% sodium alginate, and different concentrations (0%, 10%, 20%, 30%, 50%) of matrix gel. The specific results are presented as curves showing the changes in storage modulus G' and loss modulus G" of the multi-component hydrogel material system as a function of temperature / time.

[0189] like Figure 2As shown in Figure A, the Tgel of 3.75% gelatin and 1% sodium alginate in the material system without a matrix adhesive is approximately 18.5°C. Interestingly, no Tgel appears when the matrix adhesive is added. Throughout the test temperature range, G' is consistently higher than G" in the Tgel, indicating that the gel state of the bio-ink remains unchanged. Above 18.5°C, the G' and G" values ​​remain stable. Below the Tgel, G' increases rapidly, and the bio-ink exhibits properties similar to the gelatin / sodium alginate mixture. As the volume fraction of the matrix adhesive increases from 0% to 50%, both G' and G" increase significantly, indicating a corresponding increase in the viscosity of the bio-ink.

[0190] Figure 2 Figure B shows the curves of G' and G" changing over time (because the actual printing process is not instantaneous; it takes about 30-40 minutes for the entire 1 mL material system to be printed). The results show that under continuous testing at 15℃ for 40 minutes, G' and G" of bio-inks with different matrix adhesive concentrations only increased slightly, indicating that the bio-ink's performance is stable during printing. Finally, the rheological properties of the gelatin / sodium alginate / matrix composite bio-ink were compared with those of traditional pure matrix adhesive materials (commonly used at a concentration of 66.7%).

[0191] like Figure 2 As shown in Figure C, G' and G" of the pure matrix adhesive material remain essentially unchanged within the temperature range of 4-37℃.

[0192] In summary, a matrix gel concentration of 20%-30% is the optimal material ratio. Considering that matrix gel is a natural biomaterial isolated from the extracellular matrix of tumor cells, a higher concentration of matrix gel can ensure the biomimeticity of the tumor microenvironment. Therefore, a 30% concentration of matrix gel was ultimately chosen for model construction, and the optimal ratio of multi-component bio-ink is: 3.75% gelatin + 1% sodium alginate + 30% matrix gel.

[0193] 2.2 Comparison of the advantages of three-dimensional grid printing structures

[0194] To test the advantages of the three-dimensional mesh structure design, a 3D printed structure with a three-dimensional mesh structure was designed (corresponding to...). Figure 3 (Left column figure) and hydrogel embedding culture without structure design (corresponding to) Figure 3 Both methods (middle image) use a gelatin / sodium alginate / matrix multi-element hydrogel material system for bio-inks, with the matrix gel single-component ink system (corresponding to...) Figure 3 (The right-hand column of the middle figure) For comparison, the cell density was 1×10⁻⁶ under all three culture conditions. 6 per mL.

[0195] The structureless hydrogel embedding culture was specifically conducted as follows: The aforementioned bio-ink (containing complete culture medium, 3.75 wt% gelatin, 1 wt% sodium alginate, and 30 wt% matrix gelatin) was used, with a tumor cell density of 3 × 10⁻⁶ cells / year. 6 (pieces / mL), but instead of following the above 3D bioprinting procedure, the model was extruded by hand without a three-dimensional mesh structure.

[0196] The cultivation of the matrix gel single-component ink system is specifically as follows: a material system containing only matrix gel (i.e., without the addition of gelatin and sodium alginate compared to the above-mentioned 3D bioprinting bioink) is used as the bioink, and a three-dimensional mesh structure is prepared in accordance with the above-mentioned 3D bioprinting operation.

[0197] The culture conditions were as follows: cultured in a 37℃ carbon dioxide incubator, with the culture medium (i.e., complete culture medium) changed every 2-3 days. The culture conditions are described in section 3.

[0198] The growth of cells under different conditions was observed and recorded over 7 days, and the results are as follows: Figure 3 As shown, the tumor cells are A549.

[0199] The three-dimensional mesh structure design ensures that the diameter of each gel filament in the printed structure remains between 300-500μm, allowing tumor cells to proliferate and grow rapidly and uniformly. By day 7 of culture, a single cell can grow into a tumor sphere with a diameter of about 60μm.

[0200] In hydrogel-embedded culture structures without a three-dimensional mesh structure design, tumor cells cannot obtain nutrients and oxygen in a timely manner. Only cells at the edge of the structure can obtain nutrients in time, resulting in uneven growth and stunted proliferation of tumor cells in the center of the structure. Specifically, some cells remain in a single-cell morphology, and the cell growth rate is significantly slowed down. By day 7 of culture, the tumor spheres at the edge of the structure that grow faster have a diameter of about 50 μm.

[0201] In the matrix gel single ink system, the growth diameter of tumor spheres is only about 30 μm.

[0202] 3. In vitro culture

[0203] After 14 days of in vitro culture (cultured in a 37°C carbon dioxide incubator, with the culture medium changed every 2-3 days, i.e., complete culture medium), samples from the personalized tumor model were collected and analyzed, and compared with the parental tumor tissue.

[0204] 3.1 Characterization of Organizational Morphology

[0205] Five printed organoid models were subjected to HE staining and comparative analysis with their corresponding parental tumor tissues. Figure 4The results showed that the histological morphology of the printed models was highly similar to that of the cancerous tissue from which they originated, maintaining good consistency. Specifically, ccRCC samples (patient samples 1, 2, and 3) are adenocarcinomas originating from the renal tubules, composed of a single layer of tubular epithelial cells, while the "hollow cystic" structure of BP-Organoids (patient samples 1, 2, and 3) conforms to the typical developmental characteristics of tubular epithelial cells. chRCC (patient sample 5) originates from the distal collecting duct of the kidney, with cells distributed in sheets and densely arranged. The "solid cluster" growth state of BP-Organoids 5 also reflects the key morphological characteristics of chRCC. unRCC (patient sample 4) is an RCC that cannot be classified into a specific subtype, usually accompanied by unidentified cell types and mucus production, while BP-Organoids 4 also exhibits a completely different "vacuolar" tissue structure.

[0206] Furthermore, Brouier et al. pointed out that in traditional organoid culture methods, the success rate of organoid construction largely depends on the proliferation index (Ki67 positivity rate) of the parental tumor tissue; organoid models can only be successfully constructed when the Ki67 positivity rate is greater than 5%. However, the results of this application show that organoids can still be printed in vitro in 5 RCC patients even with a low Ki67 positivity rate in their parental tumor tissue, and the cell proliferation capacity (Ki67 positivity rate) of each BP-Organoids sample is very high.

[0207] These results further validate the role of multi-element hydrogel material systems in promoting organoid growth, and also demonstrate the advantage that the success rate of printed organoid construction does not depend on the proliferative capacity of parental tumor tissue and maintains the morphological consistency of parental tissue.

[0208] 3.2 Evaluation of intratumoral and intertumoral heterogeneity

[0209] To accurately assess the expression of stem cell markers in epithelial-derived renal cell carcinoma organoids, E-cadherin staining was performed on five BP-Organoid samples. Subsequently, the distribution of CD44+ / CD133+ cell populations in different printed organoid samples was examined. Figure 5 The study found significant differences in the positive rates of these two stem cell markers across five patient samples, indicating that different patients had different stem cell subpopulations, further demonstrating tumor heterogeneity. The expression levels of CD44 varied across five different regions of BP-Organoids 1 in the same patient. Figure 6 This also demonstrates the heterogeneity within the tumor.

[0210] 3.3 Verification of the stability of genetic information

[0211] Whole-exome sequencing analysis was performed on renal cell carcinoma tissue samples from 5 patients and the corresponding printed organoid models (i.e., printed samples 1 to 5). Figure 7 By comparing the proportion of shared or unique tumor-related mutations among paired samples, a high degree of consistency was found between the printed organoid model and renal cell carcinoma tissue (the shared proportions all exceeded 90%, specifically 93.61%, 90.90%, 93.24%, 92.10%, and 93.53%, respectively). The correlation coefficient of SNV mutations among samples was calculated using the Identity By Descent (IDB) method, and a correlation diagram was plotted. The results showed that the mutational characteristics of the parental renal cell carcinoma tissue and the printed organoid model originated from a common ancestor (~97%), and there was no cross-contamination among the 5 samples (correlation coefficient less than 61%).

[0212] Twenty-three renal cell carcinoma driver genes—SETD2, BAP1, PBRM1, TCEB1, TP53, BAP1, PIK3CB, CCND1, WWTR1, MTOR, PTEN, BTG1, BLM, ERBB2, PARP1, MET, VHL, APC, MGA, ASXL2, AXIN1, CDK8, and FLT1—were screened from reported literature. Their mutation forms (divided into two groups: synonymous mutations and non-synonymous / insertion / deletion mutations) were compared and analyzed between printed organoid models and renal cell carcinoma tissues. The results showed that in printed organoid models, 95% of the renal cell carcinoma driver gene mutation characteristics were consistent with those of the parental renal cell carcinoma tissue. Synonymous mutations refer to a biological genetic phenomenon where a base mutation at a certain position in the genome results in the mutated DNA fragment encoding the same amino acid due to triplet codon degeneracy, without affecting the final protein function. Non-synonymous mutations refer to gene mutations that cause functional changes in the amino acid sequence. By summarizing the proportions of seven different base mutation types (InDel, A>T / T>A, A>C / T>G, A>G / T>C, G>C / C>G, G>T / C>A, G>A / C>T), it was found that the printed organoid models were largely consistent with the parental tumor tissue. These results indicate that the bioprinted organoid models maintained the genetic map, personalized characteristics, and renal cell carcinoma-related driver mutations of the parental tumor tissue.

[0213] Figure 8 The results show the transcriptome sequencing analysis of tumor tissue and organoid models. According to the analysis results, there is consistency between the gene expression maps of the organoid models and tumor tissues in this application.

[0214] This application embodiment integrates 3D bioprinting and organoid model culture to construct a personalized renal cell carcinoma organoid model that has the parental physiological morphological characteristics and genetic information maintenance function of patient-derived cells, thus reproducing the intratumoral and intertumoral heterogeneity of the parental tissue.

[0215] 4. High-throughput model printing

[0216] While ensuring the model retains its pathological and histological heterogeneity and parental genetic information, the three-dimensional mesh structure is digested, cells are recovered, and micro-volume renal cell carcinoma organoid models are rapidly and automatically constructed using a micro-extrusion 96-well plate monolayer square high-throughput drug screening model printing method. The high-throughput bio-ink used for high-throughput printing contains 5 wt% GelMA (methacryloyl gelatin), and the tumor cell density is 3 × 10⁻⁶ cells / day. 6 The high-throughput bio-ink concentration was 0.125%, with a density of 1 mL / mL. A 27G (TT, 0.2 mm inner diameter) ultra-low adhesion dispensing needle was used to load the ink into the printing sleeve. The ink was incubated at 10°C for 5 minutes and printed according to the computer-programmed G-code file, forming droplet-shaped structures with a maximum diameter of approximately 4.5 mm. The extrusion speed was 1 mm / s, with a 3-second printing pause between wells and a 1-second retraction. A 96-well cell culture plate was used as the receiving container. The printing platform temperature was pre-set to 10°C, and the 96-well cell culture plate was pre-treated with a gel material containing 2.5% GelMA. This pre-treatment creates a non-adhesive physical surface, aiding in maintaining the morphology of the printed structure and preventing collapse and edge liquefaction. During the base coat, add 60 μL of a gel material containing 2.5% GelMA to each well, using a light intensity of 300 mW / cm². 2 Expose to ultraviolet light for 15 seconds.

[0217] The steps for digesting and recovering cells from a three-dimensional mesh structure include:

[0218] (1) The printed structures were washed with PBS buffer, and then the structures were melted with sodium chloride lysis buffer containing 55mM sodium citrate and 20mM EDTA for 5 minutes. The tumor organoid cell clusters were collected by centrifugation at 1500 rpm for 5 minutes.

[0219] (2) Next, digest the organoid cell clusters in an incubator at 37°C for 5-10 minutes with cell digestion solution (Accutase) to digest them into a single-cell suspension. The digestion solution is an enzyme solution of collagenase IV (Type-IV collagenase) with a collagenase IV content of 0.3 mg / mL.

[0220] The use of ultra-low adhesion dispensing needles and gel material underlayment can create a non-adhesive physical surface, which helps maintain the morphology of the printed structure and prevents the structure from collapsing and edge liquefaction. These two points can ensure the structural integrity of the printed model and reduce batch differences between high-throughput models.

[0221] Figure 9 The image shown is an integrated diagram of a high-content, live / dead cell stained microstructure using laser confocal microscopy. Green represents live cells, and red represents dead cells. This diagram demonstrates the uniformity and stability of the printed model.

[0222] This embodiment addresses the scarcity of patient-derived tumor cell samples by combining high-throughput drug screening model printing technology with organoid amplification culture followed by digestion and implantation. It enables rapid mass production of uniformly sized, small-volume organoids for screening and evaluation with traditional chemotherapy / targeted drugs, revealing the drug response characteristics of heterogeneous patient samples.

[0223] 5. Application of high-throughput models

[0224] Finally, a high-throughput model was used for drug detection. Specifically, the high-throughput drug screening model was treated for 48 hours with different concentrations of tancimos, cabozantinib, everolimus, cisplatin, and epirubicin. The concentrations of tancimos, cabozantinib, cisplatin, and epirubicin were 0, 10, 100, 500, 1000, and 5000 μM; the concentrations of cabozantinib, cisplatin, and epirubicin were 0, 1, 10, 50, 100, and 200 μM; the concentrations of everolimus, cisplatin, and epirubicin were 0, 1, 10, 50, 100, and 200 μM. Cell viability was detected at time points using the CCK8 kit, and response curves for the same drug were plotted for different patients based on cell viability.

[0225] Figure 10 As shown in the results, different patient models exhibited heterogeneous responses to the same drug, demonstrating the potential for personalized drug screening. For tamsilimus, only sample 4 showed a median lethal concentration (IC50), while other samples were insensitive. No samples were sensitive to cabozantinib, while samples 3 and 5 were sensitive to everolimus. However, for the chemotherapy drug cisplatin and the antibiotic epirubicin, samples 1, 2, and 3 all showed sensitive responses.

[0226] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0227] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing an organoid model, characterized in that, The preparation method includes the following steps: using bio-ink for 3D bioprinting, culturing, and preparing organoid models; The bio-ink includes: (1) A hydrogel comprising 3wt%-4wt% gelatin, 0.5wt%-2wt% sodium alginate and 20wt%-40wt% matrix gel, and a cell culture medium; wherein the cell culture medium is an organoid culture medium; wherein the organoid culture medium contains: Advanced DMEM / F12 medium, 10mM HEPES buffer, 1% glutamine, 1×B-27 serum-free additive, 1mM MN-acetylcysteine, 500nM A83-01, 20μg / mL epidermal growth factor, 10μg / mL basic fibroblast growth factor, 10μM Y-27632, 1% P / S, 0.1% bovine serum albumin; and, (2) Target cells, wherein the cell culture medium is suitable for culturing the target cells; the target cells are tumor cells; the tumor cells are primary renal cell carcinoma cells; The steps of 3D bioprinting the organoid model satisfy the following conditions: (A) Use a 25G dispensing needle; (B) The bio-ink is kept at 12.5℃-13.5℃ for 4-6 minutes before printing; (C) Stack the code layer by layer according to the instructions in the computer-programmed G-code file; (D) The fill mode is grid fill; (E) Filler density is 20%-50%; (F) Printing speed is 15mm / s-30mm / s; (G) Extrusion pressure is 3 Bar-6 Bar; (H) Extrusion speed is 2 mm / s-5 mm / s; and, (I) The retraction distance is 0.5mm-1.2mm.

2. The method for preparing organoid models according to claim 1, characterized in that, The organoid model has a three-dimensional mesh structure with a size of (2-20) × (2-20) × (1-6) mm. 3 The number of layers ranges from 2 to 20, and the average diameter of the gel filaments is 400μm-500μm.

3. The method for preparing organoid models according to claim 1, characterized in that, The density of the target cells in the bio-ink is 1×10⁻⁶. 5 Cells / mL - 1×10 7 per mL.

4. The method for preparing an organoid model according to any one of claims 1 to 3, characterized in that, Cultivate that meets one or more of the following conditions: A) The temperature is 36℃-38℃; B) Conducted in a carbon dioxide atmosphere; C) The culture medium comprises the cell culture medium as defined in claim 1; and, D) The culture medium should be changed every 2-3 days for 12-16 days.

5. A method for constructing a drug screening model, characterized in that, The construction method includes: Organoid models were prepared using the preparation method described in any one of claims 1 to 4; and, Target cells from the organoid model were collected, 3D bioprinted, solidified, and used to prepare a drug screening model.

6. The method for constructing a drug screening model according to claim 5, characterized in that, The steps of 3D bioprinting the drug screening model satisfy one or more of the following conditions: (a) The bio-ink used is another bio-ink; the other bio-ink includes other hydrogels and the target cells, the other hydrogels include 4.5wt%-5.5wt% of methacryloyl gelatin, an initiator and the cell culture medium as defined in claim 1; (b) The bio-ink used should be kept at 12.5℃-13.5℃ for 4-6 minutes before printing; (c) Print according to the computer-programmed G-code file; (d) Use a 27G dispensing needle; (e) The drug screening model is droplet-shaped; (f) The receiving container includes a cell culture plate; (g) The extrusion speed is 0.5 mm / s - 1.5 mm / s; and, (h) The printing pause time between holes is 2-4 seconds, and the retraction time is 0.5-1.5 seconds.

7. The method for constructing a drug screening model according to claim 6, characterized in that, The construction method satisfies one or more of the following conditions: I) The initiator includes a photoinitiator; II) The initiator is used in the other bio-inks at an amount of 0.1 wt%-0.2 wt%; and, (iii) The density of the target cells in the other bio-inks is 1×10⁻⁶. 5 Cells / mL - 1×10 7 per mL.

8. The method for constructing a drug screening model according to claim 7, characterized in that, The photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

9. The method for constructing a drug screening model according to claim 6, characterized in that, The maximum surface diameter of the drug screening model is 4mm-5mm.

10. The method for constructing a drug screening model according to claim 6, characterized in that, The receiving surface of the receiving container is covered with hydrogel, which includes 2wt%-3wt% methacryloyl gelatin and cell culture medium as defined in claim 1.

11. The method for constructing a drug screening model according to any one of claims 5 to 10, characterized in that, The steps for collecting target cells from the organoid model include: The organoid model was washed, lysed, and cell clusters were collected; and, The cell clusters were digested using a cell digestion solution to prepare a cell suspension.

12. The method for constructing a drug screening model according to claim 11, characterized in that, The steps of collecting target cells from the organoid model satisfy one or more of the following conditions: a) The cleaning solution used includes PBS buffer; b) The lysis conditions include: the lysis buffer used includes sodium chloride lysis buffer, 50mM-60mM sodium citrate and 15mM-25mM EDTA, and the time is 4-6 minutes. c) The cell clusters are collected by methods including centrifugation; and, d) The digestion conditions include: the cell digestion solution contains 0.2 mg / mL to 0.4 mg / mL collagenase IV, the temperature is 35℃ to 38℃, and the time is 5 minutes to 10 minutes.

13. The method for constructing a drug screening model according to claim 12, characterized in that, Centrifugation conditions include a rotation speed of 1400 rpm to 1600 rpm and a time of 4 to 6 minutes.

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