Hydrogel, bio-ink, organoid constructed by using hydrogel, drug screening model and construction method

Abstract

The invention relates to the technical field of biotechnology or biomedical engineering, in particular to hydrogel, bio-ink, organoids constructed by using the hydrogel, a drug screening model and a construction method. The hydrogel is prepared from 3 to 4 weight percent of gelatin, 0.5 to 2 weight percent of sodium alginate, 20 to 40 weight percent of matrigel and a cell culture medium. The hydrogel is helpful for improving the bionic degree of a tumor microenvironment, can be used for preparing bio-ink containing target cells such as tumor cells, particularly can be used for preparing bio-ink of primary cells (such as primary renal cell carcinoma cells), and an organ-like model constructed by using the bio-ink under a proper 3D biological printing condition is uniform in growth and high in biocompatibility. The cell growth speed is high, the consistency with parent tissues is high, the in-vitro amplification time of the patient-derived tumor cells can be shortened, the influence of subculture factors on the cell affinity can be reduced, the drug development progress can be accelerated, and the production cost can be reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology or biomedical engineering technology, and in particular to a hydrogel, a bio-ink, an organoid constructed using the same, a drug screening model, and a construction method. Background Art

[0002] In vitro cells usually lack the ability to maintain genomic integrity, and their inherent genomic instability makes them extremely prone to genetic changes and gene damage during proliferation, leading to genetic variation and genomic evolution, which interferes 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 maintenance of it in an in vitro model that is stable, accurate, and reliable. In addition, exploring methods for constructing highly biomimetic tumor microenvironments that fully restore the parental information in the patient's body, from cell composition to tissue structure, and realizing personalized, heterogeneous tumor model manufacturing based on patient-derived tumor cells, and conducting related chemotherapy / targeted drug screening and evaluation are also scientific problems and challenges in the field of biomanufacturing. In view of this, this application is hereby filed. Summary of the Invention

[0003] Based on this, one or more embodiments of the present application provide a hydrogel, a bio-ink, an organoid constructed using the same, a drug screening model, and a construction method and application thereof, including the following technical solutions:

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

[0005] In some embodiments of the present application, the cell culture medium comprises an organoid culture medium.

[0006] One or more embodiments of the present application further 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 the present 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 cancer cells; optionally, the tumor cells include primary cells; and,

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

[0012] One or more embodiments of the present application provide a bio-printing supply, the bio-printing supply comprising:

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

[0014] (II) other hydrogels or other bio-inks, wherein:

[0015] The other hydrogel comprises 4.5 wt% to 5.5 wt% of methacryloyl gelatin, an initiator, and the cell culture medium as defined above. The other bio-ink comprises the other hydrogel and the target cells as defined above.

[0016] In some embodiments of the present application, the bioprinting supplies meet one or more of the following conditions:

[0017] Ⅰ) the initiator comprises a photoinitiator; optionally, the photoinitiator comprises lithium phenyl-2,4,6-trimethylbenzoylphosphite;

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

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

[0020] One or more embodiments of the present application provide a method for preparing an organoid model, which uses the hydrogel or the bio-ink.

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

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

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

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

[0025] (B) the bio-ink is kept at 12.5° C. to 13.5° C. for 4 to 6 minutes before printing;

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

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

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

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

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

[0031] (H) an extrusion speed of 2 mm / s to 5 mm / s; and

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

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

[0034] A) Temperature is 36-38°C;

[0035] B) carried out in a carbon dioxide atmosphere;

[0036] C) the culture medium comprises a cell culture medium as defined above; and

[0037] D) The culture medium is replaced every 2 to 3 days for 12 to 16 days.

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

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

[0040] preparing an organoid model using the preparation method; and,

[0041] The target cells in the organoid model are collected, 3D bioprinted, and solidified to prepare a drug screening model.

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

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

[0044] (b) The bio-ink used was kept at 12.5°C-13.5°C for 4-6 minutes before printing;

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

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

[0047] (e) The drug screening model is in the shape of a droplet; optionally, the maximum surface diameter is 4 mm to 5 mm;

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

[0049] (g) an extrusion speed of 0.5 mm / s to 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 the present application, the step of collecting target cells in the organoid model comprises:

[0052] Washing and lysing the organoid model, and collecting cell clusters; and,

[0053] The cell clusters are digested with a cell digestion solution to prepare a cell suspension.

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

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

[0056] b) Lysis conditions include: using a lysis buffer comprising sodium chloride lysis buffer, 50 mM-60 mM sodium citrate, and 15 mM-25 mM EDTA for 4 minutes to 6 minutes;

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

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

[0059] One or more embodiments of the present application provide a drug screening model constructed by the construction method.

[0060] One or more embodiments of the present application provide a drug detection method, which includes the steps of contacting the drug to be tested with the drug screening model, and detecting the activity of the target cells in the drug screening model after the contact.

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

[0062] (i) the drug to be tested comprises one or more of temsirolimus, cabozantinib, everolimus, cisplatin, and epirubicin;

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

[0064] (iii) the concentration of the test drug to be contacted is 0 μM-5000 μM; and

[0065] (iv) Activity detection was performed using CCK8 kit.

[0066] Compared with traditional technologies, the advantages of this application include:

[0067] The present application provides a hydrogel with an optimized formula, which helps to improve the biomimetic degree of the tumor microenvironment and can be used to prepare bio-ink containing target cells such as tumor cells, especially bio-ink that can be used to prepare primary cells (such as primary renal cell carcinoma cancer cells). The organoid model constructed with this bio-ink under suitable 3D bioprinting conditions has uniform growth, fast cell growth rate, high consistency with the parent tissue, and can reduce the in vitro expansion time of patient-derived tumor cells and reduce the impact of subculture factors on cell parentality, which helps to accelerate the process of drug development and reduce production costs. In addition, the organoid model can provide a cell source for subsequent high-throughput 3D bioprinting drug screening models. Based on this, in the construction of personalized drug screening models for clinical tumor patients, the demand for clinical samples can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0069] Figure 1Invent and design a flow chart for bioprinting personalized renal cell carcinoma organoid models;

[0070] Figure 2 Rheological properties of gelatin / sodium alginate / Matrigel multicomponent bio-inks; A. Curves of G′ and G″ varying with temperature at different material ratios; B. Curves of G′ and G″ varying with time at different material ratios; C. Curves of G′ and G″ varying with temperature for composite hydrogel materials with optimal material ratios and pure Matrigel;

[0071] Figure 3 Comparison of cell growth status 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 of E-cad, CD44, and CD133 stemness markers in different BP-Organoids; scale bar, 20 μm;

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

[0075] Figure 7 Whole-exome sequencing analysis of renal cell carcinoma tissue samples and corresponding printed organoid models from five patients; (A) Ratio of shared / unique tumor-associated mutations between printed organoid samples and tumor tissue samples; (B) Heat map of the correlation of SNV mutations between printed organoid samples and tumor tissue samples; (C) Synonymous / nonsynonymous mutation signatures of renal cell carcinoma-associated driver genes in printed organoid samples and tumor tissue samples; (D) Ratio of different exon mutation types between printed organoid samples and tumor tissue samples;

[0076] Figure 8 Transcriptome sequencing analysis results of tumor tissues and organoid models;

[0077] Figure 9 This is a multi-well integrated image of high-content live-dead staining by confocal laser scanning, where green represents live cells and red represents dead cells;

[0078] Figure 10 These are the pharmacodynamic response curves of different patient samples to different chemotherapy / targeted drugs. DETAILED DESCRIPTION

[0079] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the 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 those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0081] the term

[0082] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0083] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or 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 of 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, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0084] In this application, "plurality", "multiple", "multiple times", "multiples", 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] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0086] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0087] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0088] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0089] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

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

[0091] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0092] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, 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 included therein.

[0093] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

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

[0095] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0096] Cancer is a genetic disease, defined as a polygenic disorder caused by mutations in one or more sets of genes within a cell. Key characteristics of cancer are genomic heterogeneity and instability within the tumor. This genomic heterogeneity ultimately leads to heterogeneity in phenotype, clinicopathological features, disease progression, and treatment responses among cancer patients. This heterogeneity can be categorized as intratumor heterogeneity and intertumor heterogeneity.

[0097] Organoid technology used in traditional gel-embedded cultures can effectively maintain the heterogeneity of patient-derived tumor organoids and provide screening capabilities that directly match clinical drug outcomes, making them feasible for clinical application. However, there are currently few reports on mature technologies for culturing renal cell carcinoma organoids (RCC organoid models) that can maintain the genetic characteristics of their parents and apply them to drug testing. In addition, traditional organoid culture methods face challenges in quality control and scale-up due to their difficulty in creating complex tissue structures with biomimetic multicellular components and the need for manual operation. This leads to problems such as immature functionality, low throughput, and large batch-to-batch variability. Other technologies must be combined to achieve the requirements of high-throughput practical applications.

[0098] The embodiments of this application mainly combine 3D bioprinting technology to explore new alternative methods for the manual construction of renal cell carcinoma organoids, improve the renal cell carcinoma organoid construction technology in terms of automation and biomimetic degree, and combine the pathological subtype omics characteristics of clinical patients with the heterogeneity and parental maintenance of the next-generation sequencing technology detection model. At the same time, batch screening of chemotherapy / targeted drugs is carried out based on the high-throughput 3D bioprinting drug screening model. The drug screening results are retrospectively analyzed with the phenotypic and genotypic results of the printed renal cell carcinoma organoids to explain the heterogeneous response mechanism of different drugs and realize the advancement from a universal (one-size-fits-all) treatment method to a personalized treatment method.

[0099] The beneficial effects of the embodiments of the present application include: the present application provides a hydrogel with optimized formula, which helps to improve the biomimetic degree of the tumor microenvironment, can be used to prepare bio-ink containing target cells such as tumor cells, and in particular can be used to prepare bio-ink containing primary cells (such as primary renal cell carcinoma cancer cells). The organoid model constructed with the bio-ink under appropriate 3D bioprinting conditions has uniform growth, fast cell growth rate, high consistency with the parent tissue, can reduce the expansion time of patient-derived tumor cells in vitro and reduce the influence of subculture factors on cell parentality, which helps to accelerate the process of drug development and reduce production costs. In addition, the organoid model can provide a cell source for subsequent high-throughput 3D bioprinting drug screening models. Based on this, in the construction of personalized drug screening models for clinical tumor patients, the demand for clinical samples can be reduced. The beneficial effects of the embodiments of the present application also include: (1) combining two new technologies, 3D bioprinting and organoids, to construct a new generation of methods for constructing in vitro personalized tumor models. (2) replacing traditional animal experiments with personalized renal cell carcinoma 3D bioprinting models to avoid species specificity and shorten the experimental cycle.

[0100] The first aspect of the embodiments of the present application, provides a hydrogel comprising 3wt%-4wt% of gelatin, 0.5wt%-2wt% of sodium alginate and 20wt%-40wt% of matrigel, as well as cell culture medium.

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

[0102] The present application does not specifically limit the type of cell culture medium, and an appropriate cell culture medium can be selected according to needs. When the hydrogel is used to prepare bio-ink for bioprinting, the cell culture medium of the present application can be an organoid culture medium, including but not limited to the complete culture medium involved in the examples of the present application.

[0103] The second aspect of the embodiment of the present application , also provides a bio-ink, 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 and target cells in the present application are used together. The cell culture medium in the hydrogel is suitable for the culture of the target cells. The hydrogel with the corresponding culture medium can be matched according to the type of target cells.

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

[0108] The present application does not impose any particular limitation on the density of target cells in the bio-ink, including but not limited to controlling the cell density to 1×10 5 / mL-1×10 7 / mL, for example, 1×10 5 / mL, 2×10 5 / mL, 4×10 5 / mL, 6×10 5 / mL, 8×10 5 / mL, 1×10 6 / mL, 2×10 6 / mL, 4×10 6 / mL, 6×10 6 / mL, 8×10 6 / mL, 1×10 7 pieces / mL.

[0109] In the second aspect of the embodiments of this application, Provided is a bio-printing product, comprising:

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

[0111] (II) other hydrogels or other bio-inks, wherein:

[0112] The other hydrogel comprises 4.5 wt% to 5.5 wt% of methacryloyl gelatin, an initiator, and the cell culture medium as defined above. The other bio-ink comprises the other hydrogel and the target cells as defined above.

[0113] The present application does not particularly limit the type of initiator, including but not limited to a photoinitiator. In some examples, the photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphite.

[0114] The present application does not particularly limit the amount of initiator used, and the amount of the initiator used 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% (for example, 0.1wt%, 0.12wt%, 0.14wt%, 0.16wt%, 0.18wt%, 0.2wt%).

[0115] The present application does not impose any particular limitation on the density of the target cells in other bio-inks, including but not limited to controlling the cell density to be 1×10 5 / mL-1×10 7 / mL, for example, 1×10 5 / mL, 2×10 5 / mL, 4×10 5 / mL, 6×10 5 / mL, 8×10 5 / mL, 1×10 6 / mL, 2×10 6 / mL, 4×10 6 / mL, 6×10 6 / mL, 8×10 6 / mL, 1×10 7 pieces / mL.

[0116] The third aspect of the embodiments of the present application , provides a method for preparing an organoid model, which uses the hydrogel or the biological ink.

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

[0118] The renal cell carcinoma model constructed using the above-mentioned construction method of this application has good growth conditions, in which the tumor cells can proliferate and grow rapidly and evenly, and have high consistency with the parent tumor tissue, including morphological consistency, intra-tumor and inter-tumor heterogeneity, and stable genetic information.

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

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

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

[0122] (B) The bio-ink is kept at 12.5°C-13.5°C (for example, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5°C) for 4 minutes-6 minutes (for example, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6 minutes) before printing; the main purpose of the heat preservation is to make the extruded biofilm water gel linear state.

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

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

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

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

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

[0128] (H) an extrusion speed of 2 mm / s to 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 retraction distance is 0.5 mm to 1.2 mm (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 mm).

[0130] This application does not specifically limit the culture conditions, and appropriate culture conditions can be selected according to actual needs. In some examples of this application, the culture meets one or more of the following conditions:

[0131] A) the temperature is 36° C. to 38° C. (e.g., 36, 36.2, 36.4, 36.8, 37, 37.2, 37.4, 37.6, 37.8, 38° C.);

[0132] B) carried out in a carbon dioxide atmosphere;

[0133] C) the culture medium comprises a cell culture medium as defined above; and

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

[0135] The fourth aspect of the embodiments of the present application , providing an organoid model prepared by the preparation method.

[0136] The fifth aspect of the embodiment of the present application, provides a method for constructing a drug screening model, the method comprising:

[0137] preparing an organoid model using the preparation method; and,

[0138] The target cells in the organoid model are collected, 3D bioprinted, and solidified 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 other bio-ink as defined above;

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

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

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

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

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

[0146] (g) an extrusion speed of 0.5 mm / s to 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 seconds to 4 seconds (for example, 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 seconds 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 the present application, the step of collecting target cells in the organoid model includes:

[0149] Washing and lysing the organoid model, and collecting cell clusters; and,

[0150] The cell clusters are digested with a cell digestion solution to prepare a cell suspension.

[0151] In some examples of the present 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 for cleaning includes PBS buffer;

[0153] b) lysis conditions include: using a lysis buffer comprising sodium chloride lysis buffer, 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 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);

[0154] c) collecting the cell clusters by centrifugation; optionally, the centrifugation conditions include a rotation speed of 1400 rpm-1600 rpm (e.g., 1400, 1450, 1500, 1550, 1600 rpm) and a time of 4 minutes-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) Digestion conditions include: the cell digestion solution contains 0.2 mg / mL-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) of collagenase IV, the temperature is 35°C-38°C (e.g., 35, 35.5, 36, 36.5, 37, 37.5, 38°C), and the time is 5 minutes-10 minutes (e.g., 5, 6, 7, 8, 9, 10 minutes).

[0156] The sixth aspect of the embodiments of the present application , providing a drug screening model constructed by the construction method.

[0157] The seventh aspect of the embodiment of the present application , provides a drug detection method, which includes 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 comprises one or more of temsirolimus, cabozantinib, everolimus, cisplatin, and epirubicin;

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

[0161] (iii) the concentration of the test drug contacted is 0 μM to 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 detection was performed using CCK8 kit.

[0163] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0164] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0165] Aiming at the needs of precision medicine, this application embodiment uses 3D bioprinting technology to construct a personalized in vitro tumor organoid model and drug screening model with high affinity using patient-derived tumor cells and biomimetic matrix materials. This model is then used to evaluate the efficacy of anti-tumor drugs, in order to provide a new technical means and research platform for personalized tumor treatment. This mainly includes:

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

[0167] (2) The personalized renal cell carcinoma organoid model was subjected to tissue morphology, exome and transcriptome sequencing omics analysis to evaluate the genetic stability of its single nucleotide polymorphism mutations and insertion and deletion mutations. The expression of tumor driver genes was also compared to identify its parental nature and ability to maintain intra-tumor and inter-tumor heterogeneity.

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

[0169] Combine Figure 1 , explain the embodiment scheme of this application:

[0170] 1. Tissue samples and cells

[0171] The five renal cell carcinoma patient tissue samples in this example are numbered 1 to 5, and the subsequently constructed organoid models and drug screening models are also numbered 1 to 5.

[0172] Each renal cell carcinoma tissue sample was divided into 4 parts, of which: 1 part was used to extract deoxyribonucleic acid (DNA) for exome sequencing analysis (WES), 1 part was used to extract ribonucleic acid (RNA) for transcriptome sequencing analysis (RNA sequencing, RNA-seq), and 1 part was directly used for immunohistochemistry analysis. The above three operations are used to record the biological and genetic information characteristics of the parental tumor tissue. One tissue sample was used to digest and extract tumor cells for 3D printing cross-shaped three-dimensional grid structure construction and amplification culture (i.e., operation under item 2). The steps for extracting tumor cells refer to 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 sample was mechanically processed with surgical scissors until it became 0.1-0.5 mm 3 The main steps include:

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

[0176] (2) After repeating the above washing 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, CO2 incubator for 2-3 hours; the digestion solution is: collagenase IV (Type-IV collagenase) solution with a collagenase IV content of 0.3 mg / mL;

[0177] (3) After digestion, increase the volume of the digested product to 15 mL with ice-cold DMEM / F12 medium, filter with a 70 μm filter, and then add ice-cold DMEM / F12 medium to 50 mL;

[0178] (4) Centrifuge at 1500 rpm for 5 minutes, set the centrifuge temperature to 8°C, 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°C, resuspend the cells in complete culture medium, and count them using a cell counter (Countstar) for the next experiment.

[0180] The complete culture medium used in this application contains: Advanced DMEM / F12 medium, 10mM HEPES buffer, 1% glutamine, 1×B-27 serum-free supplement, 1mM N-acetylcysteine (N-AcetyL-L-cysteine), 500nM A83-01 (transforming growth factor β kinase type 1 receptor inhibitor), 20μg / mL epidermal growth factor (EpidermaLGrowthFactor, abbreviated as EGF) + 10μg / mL basic fibroblast growth factor (basic fibrobLast growthfactor, abbreviated as bFGF), 10μM Y-27632 (ROCK inhibitor), 1% P / S, and 0.1% bovine serum albumin (BSA).

[0181] 2. Printing of organoid models (i.e., three-dimensional grid structures)

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

[0183] A 3mL syringe was used to draw up the bio-ink, equipped with a 25G (normal, inner diameter 0.26mm) dispensing needle, and loaded into the printing sleeve of a desktop extrusion-type 3D bioprinter (SUNP BIOMAKER). The ink was kept at 15°C for 5 minutes to allow the extruded bio-ink to form a straight gel state. Printing was performed under the condition that the bio-ink was in a smooth and straight gel state determined by micro-extrusion. The printing path was stacked layer by layer according to the G-code file instructions programmed by the computer; filling mode: grid filling; filling density: 30%; printing speed: 20mm / s; extrusion pressure: 5Bar; extrusion speed: 4mm / s; retraction distance: 0.6mm. The final printed hydrogel three-dimensional grid structure had a size of 10×10×2mm 3 , a total of six layers, the average diameter of the gel filaments is 500μm.

[0184] 2.1 Bio-ink Optimization

[0185] This application uses gelatin / sodium alginate / matrigel multi-component bio-ink for printing and constructing in vitro personalized 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-gel state during high-concentration gelatin printing will cause primary cell death. Therefore, a low-concentration gelatin of 3.75wt% was selected to reduce the viscosity of the bio-ink and the shear stress felt by the cells during printing.

[0187] The printing concentration of sodium alginate is usually 1wt%, but due to its certain biological inertness, it cannot provide biological sites for cell growth to adhere, resulting in the inability of cells to attach and spread, and some adherent cells may even undergo anoikis and apoptosis.

[0188] This application adds Matrigel to gelatin and sodium alginate to create a more biomimetic extracellular matrix microenvironment. Because there are few reports on Matrigel mixed printing, to clarify the appropriate Matrigel concentration, we conducted rheological analysis on a multicomponent hydrogel material system containing 3.75wt% gelatin, 1wt% sodium alginate, and varying Matrigel concentrations (0%, 10%, 20%, 30%, and 50%). The results are presented as temperature / time curves showing the storage modulus G' and loss modulus G" of the multicomponent hydrogel material system.

[0189] like Figure 2As shown in Figure A, when there is no matrix gel in the material system, the Tgel of 3.75% gelatin and 1% sodium alginate is approximately 18.5°C. Interestingly, when matrix gel is added, no Tgel appears. Throughout the test temperature range, G' is higher than G", indicating that the gel state of the bio-ink remains unchanged. When the temperature is above 18.5°C, the G' and G" values remain stable. When the temperature is below Tgel, G' increases rapidly, and the bio-ink exhibits properties similar to those of the gelatin / sodium alginate mixture. As the volume fraction of matrix gel increases from 0% to 50%, both G' and G" increase significantly, indicating that the viscosity of the corresponding bio-ink increases accordingly.

[0190] Figure 2 Figure B shows the curves of G' and G" changing with time (because the actual printing process is not completed instantly, it takes about 30-40 minutes to complete the printing of 1mL of material system). The results show that when the temperature is continuously tested at 15°C for 40 minutes, the G' and G" of the bio-inks with different Matrigel concentrations only increase slightly, indicating that the performance of the bio-ink is stable during printing. Finally, the rheological properties of the gelatin / sodium alginate / Matrigel composite bio-ink were compared with those of traditional pure Matrigel material (commonly used at a concentration of 66.7%).

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

[0192] In summary, a 20%-30% Matrigel ratio is the optimal material ratio. Considering Matrigel is a natural biomaterial isolated from the tumor extracellular matrix, a higher concentration can ensure a biomimetic effect on the tumor microenvironment. Therefore, a 30% Matrigel concentration was ultimately selected for model construction, resulting in an optimal multicomponent bioink ratio of: 3.75% gelatin + 1% sodium alginate + 30% Matrigel.

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

[0194] In order to test the advantages of the three-dimensional grid structure design, a 3D printed structure with a three-dimensional grid structure was designed (corresponding to Figure 3 Left column) and hydrogel embedding culture without structure design (corresponding Figure 3 Middle Figure) In both methods, the bio-ink used is gelatin / sodium alginate / matrigel multicomponent hydrogel system, and the matrix gel single component ink system (corresponding to Figure 3 For comparison, the cell density under the three culture conditions was 1×10 6 / mL. Among them:

[0195] The specific method of hydrogel embedding culture without structure design is as follows: the above-mentioned bio-ink (containing complete culture medium, 3.75wt% gelatin, 1wt% sodium alginate and 30wt% matrix gel, with a tumor cell density of 3×10 6 / mL), but instead of performing 3D printing according to the above-mentioned 3D bioprinting operation, a model without a three-dimensional grid structure was extruded by hand.

[0196] The specific cultivation of the single-component matrix gel ink system is as follows: a material system containing only matrix gel (i.e., gelatin and sodium alginate are not added compared to the above-mentioned 3D bioprinting bio-ink) is used as the bio-ink, and a three-dimensional grid structure is prepared by referring to the above-mentioned 3D bioprinting operation.

[0197] The culture conditions are: culture in a 37°C carbon dioxide incubator, and the culture medium (i.e., complete culture medium) is replaced every 2-3 days. The culture conditions are described in item 3.

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

[0199] The three-dimensional grid structure design can ensure that the diameter of each gel filament in the printed structure remains between 300-500μm, so that tumor cells can proliferate and grow rapidly and evenly. By the seventh day 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 grid 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 a timely manner, resulting in uneven growth and stagnation of tumor cell proliferation in the center of the structure. Specifically, some cells still exist in the form of single cells, and the cell growth rate slows down significantly. By the seventh day of culture, the diameter of the tumor spheres growing faster at the edge of the structure is about 50μm.

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

[0202] 3. In vitro culture

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

[0204] 3.1 Characterization of tissue morphology consistency

[0205] Five printed organoid models were compared with their corresponding parent tumor tissues by HE staining and comparative analysis ( Figure 4), the results showed that the histological morphological characteristics of the printed model were highly similar to the cancer tissue from which it was derived, maintaining good consistency. Specifically, the ccRCC sample (patient samples 1, 2, 3) is an adenocarcinoma derived from the renal tubules, composed of a single layer of epithelial cells growing in a luminal manner, and the "hollow cystic cavity" structure of BP-Organoids (patient samples 1, 2, 3) conforms to the typical developmental characteristics of luminal epithelial cells. chRCC (patient sample 5) originates from the distal collecting duct of the kidney, and the cells are distributed in sheets and densely arranged. The "solid cluster"-like 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, and BP-Organoids 4 also presents a completely different "vacuolar" tissue structure.

[0206] In addition, Broutier et al. pointed out that in traditional organoid culture methods, the success rate of organoid construction depends largely on the proliferation index of the parent tumor tissue (Ki67 positivity rate). Only when the Ki67 positivity rate is greater than 5% can the organoid model be successfully constructed. However, the research results of this application show that in vitro printing of organoids can still be achieved in 5 RCC patients with low Ki67 positivity rates in parental tumor tissue, and the proliferation capacity of cells in each BP-Organoids sample is very high (Ki67 positivity rate).

[0207] These results once again verified the promoting effect of the multi-component hydrogel material system on the growth of organoids, and also demonstrated that the success rate of printed organoid construction does not have to rely on the proliferation ability of the parent tumor tissue and maintains the advantage of the consistency of the parent tissue morphology.

[0208] 3.2 Evaluation of intratumor and intertumor heterogeneity

[0209] In order to accurately evaluate the expression of stem cell markers in epithelial renal cell carcinoma organoids, E-cad staining was performed on 5 BP-Organoids samples. The distribution of CD44+ / CD133+ cell populations in different printed sample organoids was then detected ( Figure 5 ), found that the positive rates of these two stem cell markers in the samples of 5 patients were significantly different, that is, different patients have different stem cell subpopulations, which once again shows the heterogeneity between tumors. The difference in the expression of CD44 in 5 different regions of BP-Organoids 1 in the same patient ( Figure 6 ) also demonstrated intratumor heterogeneity.

[0210] 3.3 Verification of genetic information stability

[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 shared or unique proportions of tumor-associated mutations between paired samples, it was found that the printed organoid model and renal cell carcinoma tissue maintained a high degree of consistency (the shared proportions were all over 90%, namely 93.61%, 90.90%, 93.24%, 92.10%, and 93.53%, respectively). The SNV mutation correlation coefficient between samples was calculated based on the IDB (Identity By Descent) method and an association diagram was drawn. The results showed that the mutation 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 between the five samples (correlation coefficient less than 61%).

[0212] From the published literature, 23 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 and their mutation patterns (divided into two groups, synonymous mutations and nonsynonymous / indel mutations) were compared between the printed organoid model and renal cell carcinoma tissue. The results showed that 95% of the renal cell carcinoma driver gene mutations in the printed organoid model were consistent with those in the parental renal cell carcinoma tissue. Synonymous mutations refer to the biological genetic phenomenon in which, after a base mutation at a certain position in the genome, the degeneracy of the triplet codon results in the mutated DNA fragment still encoding the same amino acid, without affecting the final protein function. Nonsynonymous mutations refer to gene mutations that can cause functional amino acid sequence changes. 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, and G>A / C>T) were summarized, and it was found that the printed organoid model was basically consistent with the parental tumor tissue. These results indicate that the bioprinted organoid model maintains the genetic map, personalized characteristics, and renal cell carcinoma-related driver mutations of the parental tumor tissue.

[0213] Figure 8 The following are the transcriptome sequencing analysis results of tumor tissue and organoid model. According to the analysis results, there is consistency in the gene expression profiles of the organoid model and tumor tissue of the present application.

[0214] The embodiments of the present application integrate 3D bioprinting and organoid model culture to construct a personalized renal cell carcinoma organoid model that has the same physiological morphological characteristics and genetic information maintenance function as patient-derived cells, and reproduces the intratumoral and intertumor heterogeneity of the parental tissue.

[0215] 4. High-throughput model printing

[0216] While ensuring the model maintains its pathological histological heterogeneity and genetic information parentage, the three-dimensional grid structure is digested and the cells are recovered. A micro-volume renal cell carcinoma organoid model is quickly and automatically constructed by micro-extrusion printing of a single-layer square high-throughput drug screening model in a 96-well plate. The high-throughput bio-ink used for high-throughput printing contains 5wt% GelMA (methacryloyl gelatin) and a tumor cell density of 3×10 6 The high-throughput bio-ink was drawn up using a 1mL glass syringe and loaded into the print cartridge using a 27G (TT, 0.2mm inner diameter) ultra-low adhesion dispensing needle. The ink was incubated at 10°C for 5 minutes and then printed according to the computer-programmed G-code file. The ink was printed as droplet-shaped structures with a maximum surface diameter of approximately 4.5mm. The extrusion speed was 1mm / s, with a 3-second pause between wells and a 1-second retraction. The receiving container was a 96-well cell culture plate. The printing platform temperature was set to 10°C and the plate was pre-treated with a 2.5% GelMA-containing gel. This pre-treatment creates a non-adherent physical surface, which helps maintain the morphology of the printed structure and prevents structure collapse and edge liquefaction. When laying the bottom, add 60 μL of gel material containing 2.5% GelMA to each well and use a light intensity of 300 mW / cm 2 UV light for 15 seconds.

[0217] The steps of digesting the three-dimensional grid structure and recovering cells include:

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

[0219] (2) Then, the organoid cell clusters were digested with cell digestion solution (Accutase) in a 37°C incubator for 5-10 minutes to digest them into single-cell suspensions, wherein the digestion solution was a collagenase IV (Type-IV collagenase) enzyme solution with a collagenase IV content of 0.3 mg / mL.

[0220] The use of ultra-low adhesion dispensing needles and the gel material base treatment method can produce a non-adherent physical surface, helping to maintain the morphology of the printed structure and prevent structure collapse and edge liquefaction. These two points can ensure the structural integrity of the printed model and reduce batch variability between high-throughput models.

[0221] Figure 9 This image shows a multi-well integrated image of high-content live-dead staining using confocal laser scanning. Live cells are shown in green, while dead cells are shown in red. This image demonstrates the uniformity and stability of the printed model.

[0222] This example addresses the scarcity of patient-derived tumor cell samples through a high-throughput drug screening model printing process, combined with organoid expansion and culture followed by digestion and implantation. This allows for rapid batch production of uniformly sized, small-volume organoids, enabling traditional chemotherapy / targeted drug screening and evaluation, revealing heterogeneous drug response characteristics across patient samples.

[0223] 5. Application of high-throughput models

[0224] Finally, the high-throughput model was used for drug testing. Specifically, the high-throughput drug screening model was treated with varying concentrations of tansilimos, cabozantinib, everolimus, cisplatin, and epirubicin for 48 hours. Tansilimos concentrations were 0, 10, 100, 500, 1000, and 5000 μM; cabozantinib concentrations were 0, 1, 10, 50, 100, and 200 μM; everolimus concentrations were 0, 5, 10, 25, 50, and 100 μM; cisplatin concentrations were 0, 5, 10, 50, 100, and 250 μM; and epirubicin concentrations were 0, 1, 10, 50, 100, and 200 μM. Cell activity was detected using the CCK8 kit at time points, and response curves of different patients to the same drug were drawn based on cell survival rates.

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

[0226] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes 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 the description in this specification.

[0227] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A hydrogel, characterized in that The hydrogel comprises 3wt%-4wt% of gelatin, 0.5wt%-2wt% of sodium alginate, 20wt%-40wt% of matrigel, and a cell culture medium; optionally, the cell culture medium comprises an organoid culture medium.

2. A bio-ink, characterized in that: The bio-ink comprises: (1) The hydrogel according to claim 1, and (2) Target cells, wherein the cell culture medium is suitable for culturing the target cells.

3. The bio-ink according to claim 2, characterized in that The bio-ink satisfies one or more of the following conditions: 1) the target cells include tumor cells; optionally, the tumor cells include renal cell carcinoma cancer cells; optionally, the tumor cells include primary cells; and, 2) The density of the target cells in the bio-ink is 1×10 5 / mL-1×10 7 pieces / mL.

4. A bioprinting product, characterized in that: The bioprinting supplies include: (I) the hydrogel according to claim 1 or the bio-ink according to any one of claims 2 to 3; and (II) other hydrogels or other bio-inks, wherein: The other hydrogel comprises 4.5wt%-5.5wt% of methacryloyl gelatin, an initiator, and the cell culture medium defined in claim 1 , and the other bio-ink comprises the other hydrogel and the target cells defined in any one of claims 2 to 3 .

5. The bioprinting product according to claim 4, characterized in that: The bio-printing supplies meet one or more of the following conditions: Ⅰ) the initiator comprises a photoinitiator; optionally, the photoinitiator comprises lithium phenyl-2,4,6-trimethylbenzoylphosphite; II) the amount of the initiator in the other bio-ink is 0.1 wt%-0.2 wt%; and, III) The density of the target cells in the other bio-ink is 1×10 5 / mL-1×10 7 pieces / mL.

6. A method for preparing an organoid model, characterized in that: The preparation method uses the hydrogel according to claim 1 or the bio-ink according to any one of claims 2 to 3; Optionally, the preparation method comprises the following steps: performing 3D bioprinting using the bio-ink, culturing, and preparing an organoid model; Optionally, the organoid model has a three-dimensional grid structure with a size of (2-20)×(2-20)×(1-6) mm 3 The number of layers is 2 to 20, and the average diameter of the gel filaments is 400 μm to 500 μm.

7. The method for preparing an organoid model according to claim 6, wherein: The step of 3D bioprinting the organoid model satisfies one or more of the following conditions: (A) Using a 25G dispensing needle; (B) the bio-ink is kept at 12.5° C. to 13.5° C. for 4 to 6 minutes before printing; (C) stacking layer by layer according to the G-code file instructions programmed by the computer; (D) The filling mode is grid filling; (E) Filling density is 20%-50%; (F) Printing speed is 15mm / s-30mm / s; (G) Extrusion pressure is 3Bar-6Bar; (H) an extrusion speed of 2 mm / s to 5 mm / s; and (I) The retraction distance is 0.5mm-1.2mm.

8. The method for preparing an organoid model according to any one of claims 6 to 7, characterized in that: Cultivate one or more of the following conditions: A) Temperature is 36-38°C; B) carried out in a carbon dioxide atmosphere; C) the culture medium comprises the cell culture medium defined in claim 1; and D) The culture medium is replaced every 2 to 3 days for 12 to 16 days.

9. An organoid model, characterized in that Prepared by the preparation method according to any one of claims 6 to 8.

10. A method for constructing a drug screening model, characterized in that: The construction method comprises: Preparing an organoid model using the preparation method according to any one of claims 6 to 8; and The target cells in the organoid model are collected, 3D bioprinted, and solidified to prepare a drug screening model.

11. The method for constructing a drug screening model according to claim 10, wherein: The step of 3D bioprinting the drug screening model satisfies one or more of the following conditions: (a) The bio-ink used is any one of the bio-inks defined in claims 4 to 5; (b) The bio-ink used was kept at 12.5°C-13.5°C for 4-6 minutes before printing; (c) Printing according to the G-code file programmed by the computer; (d) Use a 27G dispensing needle; (e) The drug screening model is in the shape of a droplet; optionally, the maximum surface diameter is 4 mm to 5 mm; (f) The receiving container comprises a cell culture plate; optionally, the receiving surface of the receiving container is paved with a hydrogel, wherein the paved hydrogel comprises 2 wt% to 3 wt% of methacryloyl gelatin and the cell culture medium defined in claim 1; (g) an extrusion speed of 0.5 mm / s to 1.5 mm / s; and (h) The printing pause time between holes is 2 seconds to 4 seconds, and the retraction time is 0.5 seconds to 1.5 seconds.

12. The method for constructing a drug screening model according to any one of claims 10 to 11, characterized in that: The steps of collecting target cells in the organoid model include: Washing and lysing the organoid model, and collecting cell clusters; and, digesting the cell clusters with a cell digestion solution to prepare a cell suspension; Optionally, the step of collecting target cells in the organoid model satisfies one or more of the following conditions: a) The cleaning solution used for cleaning includes PBS buffer; b) Lysis conditions include: using a lysis buffer comprising sodium chloride lysis buffer, 50 mM-60 mM sodium citrate, and 15 mM-25 mM EDTA for 4 minutes to 6 minutes; c) collecting the cell clusters by centrifugation; optionally, the centrifugation conditions include a rotation speed of 1400 rpm to 1600 rpm and a time of 4 minutes to 6 minutes; and d) Digestion conditions include: the cell digestion solution contains 0.2 mg / mL-0.4 mg / mL collagenase IV, the temperature is 35° C.-38° C., and the digestion time is 5 minutes-10 minutes.

13. A drug screening model, characterized in that: Constructed by the construction method according to any one of claims 10 to 12.

14. A method for detecting a drug, characterized in that: The detection method comprises the steps of contacting the drug to be tested with the drug screening model according to claim 13, and detecting the activity of the target cells in the drug screening model after the contact.

15. The drug screening method according to claim 14, characterized in that: The detection method meets one or more of the following conditions: (i) the drug to be tested comprises one or more of temsirolimus, cabozantinib, everolimus, cisplatin, and epirubicin; (ii) The contact time is 40 hours to 55 hours; (iii) the concentration of the test drug to be contacted is 0 μM-5000 μM; and (iv) Activity detection was performed using CCK8 kit.

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