Composition for constructing complex PNET organ-like model, model construction method and application

Abstract

The invention discloses a composition for constructing a complex PNET organ-like model, a model construction method and application. The composition comprises a penicillin-streptomycin double antibody solution, GlutaMax, a serum-free supplement, a Wnt signal channel activator, a human recombinant Noggin protein, a human recombinant FGF10 protein, HEPES, N-acetyl-L-cysteine, gastrin, nicotinamide, an adenylate cyclase activator, an ALK inhibitor, a ROCK inhibitor and a p38MAPK inhibitor. The composition provided by the invention comprises a plurality of growth factors required for vascularization growth of PNET organs, and can meet the requirements of PNET organ cells on nutrient substances and regulation and control substances in a complex structure growth process; in the culture process, a complex PNET organ-like biological model which is closer to the real tumor microenvironment of a human body and is more simulated and more comprehensive is developed, and the PNET organ-like biological model has wider and more important application value in the aspects of clinical diagnosis of PNET diseases, drug research, development and screening, precision medical treatment and the like.

Description

Technical Field

[0001] This application relates to the field of cell engineering technology, and in particular to a composition, model building method and application for constructing complex PNET organoid models. Background Technology

[0002] Pancreatic neuroendocrine tumors (PNETs) belong to the category of neuroendocrine tumors and are a group of heterogeneous tumors originating from peptidergic neurons and neuroendocrine cells, specifically adeno-neuronal tumors. Pancreatic neuroendocrine tumors are a rare disease, with an incidence of approximately 0.3 per 100,000, accounting for less than 10% of pancreatic tumors. However, recent studies have found that they are prone to distant metastasis, with only 14% remaining confined to the pancreas. Local metastasis occurs in 22% of cases, and distant metastasis reaches as high as 64%. Based on their hormone-secreting capacity, PNETs can be divided into "functional" and "non-functional" categories. Functional tumors secrete large amounts of hormones, producing corresponding symptoms; non-functional tumors do not produce or cannot produce sufficient amounts of hormones, causing significant symptoms. Therefore, they are difficult to detect early and are often diagnosed only after metastasis. Non-functional pancreatic neuroendocrine tumors account for approximately 70%-80% of all cases. Furthermore, the etiology of pancreatic neuroendocrine tumors is still unclear, the genetic molecular mechanisms are unknown, and there are no specific therapeutic drugs.

[0003] Organoids are cell clusters formed by 3D in vitro culture of stem cells, which utilize the self-organizing properties of stem cells. They can simulate the structure and function of tissues in vivo to the greatest extent and can be stably passaged for a long time, providing a new model for exploring the pathogenesis of the source organ and screening drugs for tumors.

[0004] Existing organoid models used for PNET research have certain limitations. For example, most PNET organoids lack vascularized structures, are prone to central necrosis due to oxygen deficiency and increased metabolic waste, and lack immune components and intercellular interactions, making them unable to fully simulate the human physiological environment. Furthermore, the absence of cells from the tumor microenvironment in organoid culture also affects tumor behavior, thus hindering a more realistic simulation of the tumor microenvironment within the human body. Therefore, there is an urgent need to establish a highly realistic PNET biological model for research on its pathogenesis and drug screening, which has significant scientific and clinical implications for elucidating the pathology of this disease and optimizing treatment plans. Summary of the Invention

[0005] This application provides a composition, a method for constructing complex PNET organoid models, and an application thereof. The complex PNET organoid model constructed using the composition of this application is structurally a simulated complex tumor model, which solves the problem of existing PNET organoid models being too simplistic and having poor simulation accuracy.

[0006] In a first aspect, embodiments of this application provide a composition for constructing complex PNET organ-like models, comprising the following components:

[0007] Based on the volume concentration of the final volume of the composition,

[0008] Penicillin-streptomycin dual antibiotic solution, 1% vol / vol to 5% vol / vol;

[0009] GlutaMax, 1%vol / vol~10%vol / vol;

[0010] Serum-free supplements, including B-27 at 0.5% vol / vol to 10% vol / vol and N-2 at 1% vol / vol to 10% vol / vol;

[0011] Wnt signaling pathway activator, 1%–20% vol / vol;

[0012] Based on the final volume mass concentration of the composition,

[0013] Human recombinant Noggin protein, 10 μg / L~500 μg / L;

[0014] Human recombinant FGF10 protein, 50 ng / mL to 150 ng / mL;

[0015] Based on the final volume molar concentration of the above composition,

[0016] HEPES, 1 mmol / L to 20 mmol / L;

[0017] N-acetyl-L-cysteine, 0.2 μmol / L~5 μmol / L;

[0018] Gastrin, 0.5 μmol / L~20 μmol / L;

[0019] Nicotinamide, 0.1 mmol / L to 20 mmol / L;

[0020] Adenylate cyclase activator, 2 μmol / L~20 μmol / L;

[0021] ALK inhibitors, 0.1 μmol / L~10 μmol / L;

[0022] ROCK inhibitors, 0.1 μmol / L~30 μmol / L;

[0023] p38MAPK inhibitor, 0.05 μmol / L~10 μmol / L.

[0024] For example, in some embodiments, the volume concentration of the penicillin-streptomycin bispecific antibody solution is 1% vol / vol to 5% vol / vol. For instance, the volume concentration of the penicillin-streptomycin bispecific antibody solution can be 1, 1.2, 1.7, 2.2, 2.8, 3.4, 3.9, 4.1, 4.3, 4.8, 5, or any value within the range of any two of these values; the volume concentration of GlutaMax is 1% vol / vol to 10% vol / vol. For instance, the volume concentration of GlutaMax can be 1, 1.3, 2.6, 3.3, 4.2, 5.6, 6.3, 7.8, 8.9, 9.5, 10, or any value within the range of any two of these values. Serum-free supplements include B-27 at concentrations of 0.5% vol / vol to 10% vol / vol, for example, B-27 volume concentrations can be 0.5, 0.8, 1.6, 2.2, 3.2, 4.5, 5.2, 6.1, 6.8, 7.7, 8.9, 9.4, 10, or any combination thereof; for example, N-2 volume concentrations can be 1, 1.1, 2.1, 3.2, 4.3, 4.7, 5.1, 6.2, 6.8, 7.9, 8.6, 9.3, 10, or any combination thereof; and Wnt signaling pathway activators at volume concentrations of 1% to 20% vol / vol, for example, Wn The volumetric concentration of the t signaling pathway activator can be 1, 4, 6, 8, 11, 12, 13, 15, 18, 20, or any combination of these values; the mass concentration of recombinant human Noggin protein is 10 μg / L to 500 μg / L, for example, the mass concentration of recombinant human Noggin protein can be 10, 32, 71, 121, 165, 191, 255, 298, 348, 392, 420, 487, 500, or any combination of these values; the mass concentration of recombinant human FGF10 protein is 50 ng / mL to 150 ng / mL, for example, the mass concentration of recombinant human FGF10 protein can be 50, 53, 50 ... Values ​​in the range of 62, 77, 78, 88, 99, 111, 116, 123, 141, 145, 150, or any combination of these values; the molar concentration of HEPES is 1 mmol / L to 20 mmol / L, for example, the molar concentration of HEPES can be 1, 2, 3, 5, 7, 9, 10, 12, 14, 16, 18, 20, or any combination of these values; the molar concentration of N-acetyl-L-cysteine ​​is 0.2 μmol / L to 5 μmol / L, for example, the molar concentration of N-acetyl-L-cysteine ​​can be 0.2, 0.6, 0.7, 1.1, 1.9, 2.7, 3.1, 3.5, 4.0, 4.3, 5, or any two of these values ​​within a range; the molar concentration of gastrin is 0.5 μmol / L to 20 μmol / L, and the molar concentration of gastrin can be 0.5, 2.1, 3.3, 4.9, 6.1, 8.1, 9.8, 11.5, 14.3, 15.2, 18.0, 19.1, or any two of these values ​​within a range; the molar concentration of nicotinamide is 0.1 mmol / L to 20 mmol / L, for example, nicotinamide... The molar concentration can be 0.1, 0.9, 3, 5, 6, 9, 10, 13, 16, 20, or any combination of these values; the molar concentration of the adenylate cyclase activator is 2 μmol / L to 20 μmol / L, for example, the molar concentration of the adenylate cyclase activator can be 2, 3, 5, 6, 8, 9, 12, 13, 14, 17, 18, 19, 20, or any combination of these values; the molar concentration of the ALK inhibitor... The concentration ranges from 0.1 μmol / L to 10 μmol / L. For example, the molar concentration of ALK inhibitors can be 0.1, 1.0, 1.6, 2.8, 2.9, 4.3, 5.2, 5.9, 7.3, 8.6, 9.2, 10, or any combination of these values. The molar concentration of ROCK inhibitors ranges from 0.1 μmol / L to 30 μmol / L. For example, the molar concentration of ROCK inhibitors can be 0.1, 1, 4, 7, 9, 1... 2, 14, 18, 20, 22, 26, 28, 30, or any combination of these values; the molar concentration of the p38MAPK inhibitor is 0.05 μmol / L to 10 μmol / L. For example, the molar concentration of the p38MAPK inhibitor can be 0.05, 0.4, 1.5, 2.7, 3.2, 4.4, 5.4, 6.0, 6.7, 8.1, 8.8, 9.8, 10, or any combination of these values.

[0025] Based on the above embodiments, the composition for constructing complex PNET organoid models provided in this application, by adding the components of the above formulation to the primary culture system during the model construction process, includes a variety of growth factors required for the vascularization growth of PNET organoids, and regulates the parameters of various components in the culture medium within the above range, so that the various factors coordinate and cooperate to meet the nutritional and regulatory needs of PNET organoid cells during the growth of complex structures, thereby helping to construct a fully developed and healthy 3D model structure, and further promoting the development of PNET organoid cells into a more realistic and comprehensive complex PNET organoid biological model that is closer to the real tumor microenvironment in humans during the culture process. It has a wider and more important application value in the clinical diagnosis, drug development and screening, and precision medicine of PNET diseases.

[0026] In some embodiments, the aforementioned Wnt signaling pathway activators include one or more of R-spondin-1, BML-284, SM-04554, CHIR-99021, HLY78, or SK-3530 dihydrochloride. BML-284, also known as CID11210285 hydrochloride, 4-N-(1,3-benzodiazol-5-methylene)-6-(3-methoxyphenyl)-2,4-diaminopyrimidine, CAS No.: 853220-52-7, molecular formula C 19 H 18 N4O3; SM-04554, also known as Dalosirvat, 1-(2,3-dihydro-1,4-benzodioxane-6-yl)-4-phenyl-1,4-butanedione; CHIR-99021, also known as Laduviglusib, CT99021, 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarboxynitrile, CAS No.: 252917-06-9, molecular formula C 22 H 18 Cl2N8; HLY78, Chinese name: 4-ethyl-5-methyl-5,6-dihydro-[1,3]dioxabenzo[4,5-j]phenanthridine, CAS number: 854847-61-3, molecular formula: C 17 H 17 NO2. In some embodiments, the adenylate cyclase activator is selected from Forskolin and / or NKH477. Forskolin, also known as hairy throat extract, HL 362, or Forskolin, CAS No.: 66575-29-9, molecular formula C 22 H 34 O7; NKH477, also known as Dafalcocin, N,N-dimethyl-(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-5-(acetoxy)-3-vinyldodecylhydro-10,10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-1H-naphthalene[2,1-b]pyran-6-yl ester β-alanine hydrochloride, CAS No.: 138605-00-2, molecular formula C 27 H 44 ClNO8. In some embodiments, the above-mentioned ALK inhibitor is selected from one or more of A83-01, SB-505124, or TP-0184. A83-01, Chinese name 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide, CAS number: 909910-43-6, molecular formula C 25 H19 N5S; SB-505124, Chinese name: 2-[4-(1,3-benzodioxo-5-yl)-2-(tert-butyl)-1H-imidazol-5-yl]-6-methylpyridine, CAS number: 694433-59-5, molecular formula: C 20 H 21 N3O2; TP-0184, also known as Itacnosertib, (6E)-6-(2,2'-bipyridine-3-imino)-N-[3-methylamino-4-(4-methyl-1-piperazinyl)phenyl]-1,6-dihydro-2-pyrimidinylamine, CAS No.: 1628870-27-8, molecular formula C 26 H 28 N8O. In some embodiments, the above-mentioned ROCK inhibitor is selected from Y27632 and / or HA-1077. Y27632, also known as Y-27632 dihydrochloride, 4-[(1R)-1-aminoethyl]-N-(pyridin-4-yl)cyclohexane-1-carboxamide, CAS No.: 146986-50-7, molecular formula C 14 H 21 N3O. HA-1077, also known as AT877, fasudil, hexahydro-1-(5-isoquinolinesulfonyl)-1H-1,4-diazazolide, CAS No.: 103745-39-7, molecular formula C 14 H 17 N3O2S. In some embodiments, the above-mentioned p38MAPK inhibitor is selected from one or more of SB202190, Adezmapimod, or Doramapimod. SB202190, Chinese name 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazol, CAS number: 152121-30-7, molecular formula C 20 H 14 FN3O; Adezmapimod, also known as SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazolium, CAS No.: 152121-47-6, molecular formula C 21 H 16 FN3OS; Doramimod, also known as BIRB 796, Damamod, 1-[2-(4-methylphenyl)-5-tert-butylpyrazole-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphth-1-yl]urea, CAS No.: 285983-48-4, molecular formula C 31 H 37N5O3. Based on the above embodiments, this application selects the above-mentioned Wnt signaling pathway activator, adenylate cyclase activator, ALK inhibitor, ROCK inhibitor, and p38MAPK inhibitor, and controls them within a certain range, so that they can act as a system of mutually influencing regulatory factors to meet the nutritional and regulatory needs of PNET organoid cells during the growth of complex structures, thereby helping to construct a fully developed and healthy 3D model structure.

[0027] In some embodiments, the composition further includes a basal culture medium, which may include Advanced DMEM / F12 medium or DMEM medium. Based on the above embodiments, by selecting the above basal culture medium for synergistic use with the composition, this application can better accommodate the material preferences of PNET organoid cells during growth, further helping to construct fully developed and healthy 3D model structures.

[0028] Secondly, embodiments of this application provide a method for constructing a complex PNET organoid model, including the step of culturing primary or passaged PNET organoids using the above-described composition.

[0029] In some embodiments, the above construction method specifically includes:

[0030] S1: Pretreatment: Prepare cell pellets from vascular endothelial cells and fibroblasts for later use; digest primary or passaged PNET organoids to obtain uniformly sized cell clusters for later use; add anti-adhesion reagent to each well of a U-shaped bottom multi-well culture plate. Use at least one of the following: anti-adhesion solution (AIMINGMED, catalog number #100-291), anti-adhesion rinsing solution (Stemcell, catalog number #07010), anti-adhesion rinsing solution (bioGenous, catalog number #E238002), and organoid anti-adhesion rinsing solution (Mogengel, catalog number #MB-0818L03L). After standing, remove the anti-adhesion reagent, add PBS buffer to rinse, and let stand. Repeat rinsing 2 to 3 times.

[0031] S2: Preparation of cell suspension: The above composition is prepared into an organoid model culture medium, and then the organoid model culture medium is mixed with endothelial cell culture medium and fibroblast culture medium to prepare a mixed culture medium; the prepared PNET organoid cell clusters, vascular endothelial cell precipitate and fibroblast precipitate are mixed to prepare a mixed cell suspension;

[0032] S3: Plating and culture. Add 3,000 to 15,000 cells to each well of the U-shaped bottom multi-well culture plate treated in S1, and then centrifuge. Inject mixed culture medium into the centrifuged U-shaped bottom multi-well culture plate and incubate for 1 to 2 days.

[0033] S4: Rotate the ball, aspirate the culture medium in each well of the U-shaped bottom multi-well culture plate, add matrix gel, and transfer the complex PNET organoid model spheroids to the flat bottom multi-well culture plate;

[0034] S5: Model construction and culture. Place a flat-bottomed multi-well culture plate in an incubator, let the gel stand still, add mixed culture medium, and place it in an incubator for static culture to obtain a complex PNET organoid model.

[0035] The method for constructing a complex PNET organoid model based on the embodiments of this application, by mixing and culturing PNET organoid cell clusters with vascular endothelial cell deposits and fibroblast deposits in a certain proportion, can simulate the real environment in which PNET tumors occur and grow in vivo, thereby developing into a simulated complex PNET organoid model consisting of PNET organoid tumor structures, fibroblast structures and microvascular structures. This model is closer to the biochemical characteristics of the original PNET tumor tissue and is more reliable and easy to operate when applied to the clinical diagnosis and drug development of PNET diseases.

[0036] In some embodiments, the mass ratio of organoid model culture medium, endothelial cell culture medium, and fibroblast culture medium in the above-mentioned mixed culture medium is 1:(0.5-1):(0.1-0.5). Based on the above embodiments, by controlling the mass ratio of each component in the mixed culture medium within a certain range, this application can more accurately meet the needs of various cells during growth and development, and further help to construct a fully developed and healthy 3D model structure.

[0037] In some embodiments, the fibroblasts include cancer-associated fibroblasts. The cell ratio of the PNET organoid cells, cancer-associated fibroblasts, and vascular endothelial cells is 1:(0.2-0.9):(0.1-0.8). Based on the above embodiments, by selecting cancer-associated fibroblasts and controlling the cell ratio of PNET organoid cells, cancer-associated fibroblasts, and vascular endothelial cells within the above range, this application can further simulate the real environment of PNET tumor occurrence and growth in vivo, thereby further helping to construct a fully developed and healthy 3D model structure.

[0038] Thirdly, embodiments of this application provide a complex PNET organ model constructed using the above-described construction method.

[0039] Fourthly, embodiments of this application provide the application of the aforementioned complex PNET organoid model in the development of drugs for pancreatic neuroendocrine tumors.

[0040] In some embodiments, the above applications include at least one of drug screening, drug sensitivity testing, drug toxicity testing, or non-drug toxicity testing for pancreatic neuroendocrine tumors.

[0041] In some embodiments, the above application includes at least the following steps:

[0042] The test drug was added to the culture vector containing the above-mentioned complex PNET organoid model, and cultured for 3-5 days. The changes in the indicators of the above-mentioned complex PNET organoid model were then detected.

[0043] In some embodiments, the aforementioned changes in indicators include at least one of the following: changes in size, integrity, activity, or inhibition rate of the complex PNET organoid model. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0045] Figure 1 This is a flowchart of the experimental process for Example 3 of this application;

[0046] Figure 2 This is a comparative diagram of the PNET in vitro culture model in Example 1 of this application, wherein... Figure 2 -(A) is a typical single PNET organoid bright-field image (10X magnification); Figure 2 -(B) Bright-field image (10X magnification) of a successfully constructed complex PNET organoid model;

[0047] Figure 3 This is a bright-field image record of surufatinib acting on a complex model in the drug sensitivity test of Example 2 of this application;

[0048] Figure 4 This is the result of the activity test of surufatinib on the complex model day5 in the drug sensitivity test of Example 2 of this application. Detailed Implementation

[0049] Material Specifications

[0050] Penicillin-streptomycin bispecific antibody solution (Gibco, catalog number #15140122); GlutaMax (Gibco, catalog number #35050061); B-27 (Gibco, catalog number #17504044); N-2 (Gibco, catalog number #17502048); R-spondin-1 (Gibco, catalog number #120-38); recombinant human Noggin protein (MCE, catalog number #HY-P70558); recombinant human FGF10 protein (PeproTech, catalog number #100-26); HEPES (Gibco, catalog number #15630080); N-acetyl-L-cysteine ​​(MCE, catalog number #HY-B0215); gastrin (MCE, catalog number #HY-P1097); nicotinamide (Sigma-Aldrich, catalog number #N2630); NKH477 (MCE, catalog number #HY-103193); SB-505124 (MCE, catalog number #HY-13521); HA-1077 (MCE, catalog number #HY-10341A); SB202190 (MCE, catalog number #HY-10295); DMEM medium (Gibco, catalog number #12491015); Anti-Adherence Rinsing Solution (bioGenous, catalog number #E238002); PBS buffer (Gibco, catalog number #10010023); CTG reagent (Promega, catalog number #G9682).

[0051] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0052] Example 1

[0053] This example provides a culture medium for constructing complex PNET organoid models, the components of which include:

[0054] Based on the final volume concentration of the culture medium,

[0055] Penicillin-streptomycin dual antibiotic solution, 2.3% vol / vol;

[0056] GlutaMax, 5.5% vol / vol;

[0057] Serum-free supplements, including 3.6% vol / vol of B-27 and 6.8% vol / vol of N-2;

[0058] R-spondin-1, an activator of the Wnt signaling pathway, 12% vol / vol;

[0059] Based on the final volume of the culture medium,

[0060] Human recombinant Noggin protein, 200 μg / L;

[0061] Human recombinant FGF10 protein, 75 ng / mL;

[0062] Based on the molar concentration of the final volume of the culture medium,

[0063] HEPES, 13 mmol / L;

[0064] N-acetyl-L-cysteine, 3.2 μmol / L;

[0065] Gastrin, 12 μmol / L;

[0066] Nicotinamide, 10 mmol / L;

[0067] Adenylate cyclase activator NKH477, 2 μmol / L;

[0068] ALK inhibitor SB-505124, 6 μmol / L

[0069] ROCK inhibitor HA-1077, 30 μmol / L;

[0070] p38MAPK inhibitor SB202190, 0.05 μmol / L~10 μmol / L;

[0071] The remainder is DMEM culture medium.

[0072] Example 2

[0073] This example provides a method for constructing a complex PNET organ-like model. The construction method specifically includes:

[0074] (1) Take vascular endothelial cells and cancer-related fibroblasts, revive and passage them. When the cells are in good condition and the confluence reaches about 90%, digest and centrifuge them to obtain cell pellets for later use.

[0075] (2) Take primary PNET organoids or resuscitate passaged PNET organoids. When the organoids are in good condition and 70% or more are about 70 μm to 100 μm in size, digest the organoids. When digestion is completed into uniform cell clusters, stop digestion to obtain uniform cell clusters for later use.

[0076] (3) Take a U-shaped bottom 96-well plate, add 50μL to 150μL of anti-adhesion reagent Anti-Adhesion Rinsing Solution (bioGenous, catalog number #E238002) to each well, let it stand in a clean bench for 20min, after standing, remove the anti-adhesion reagent, add 100μL of PBS buffer to each well to rinse, and let it stand for 5min to 10min. Repeat the rinsing 2 to 3 times.

[0077] (4) The organoid model culture medium prepared in Example 1 was mixed with endothelial cell culture medium and cancer-associated fibroblast culture medium at a mass ratio of 1:0.5:0.5 to prepare a mixed culture medium; the prepared PNET organoid cell clusters, vascular endothelial cell precipitates and cancer-associated fibroblast precipitates were mixed at a cell number ratio of 1:0.6:0.3 to prepare a mixed cell suspension;

[0078] (5) Add 200 μL of cell suspension, containing approximately 3,000 to 15,000 cells, to each well of the U-shaped bottom 96-well plate treated in step (3), and then centrifuge at 400g for 5 min; inject the mixed culture medium prepared in step S2 into the centrifuged U-shaped bottom 96-well plate, place it in an incubator, and incubate at 37°C and 5% CO2 for 1 to 2 days;

[0079] (6) Gently aspirate the culture medium in each well of the U-shaped 96-well plate, add 5 μL of matrix gel to each well, and gently and quickly transfer the complex PNET organoid model spheroids to the flat-bottomed multi-well culture plate.

[0080] (7) Place the flat-bottomed 96-well culture plate in a 37°C incubator and let the gel stand for 30 min. Add 100 μL of mixed culture medium to each well and place it in a 37°C incubator with 5% CO2 for static culture to obtain a complex PNET organoid model.

[0081] Example 3

[0082] This example provides a method for applying the complex PNET organoid model described in this application to the development of drugs for pancreatic neuroendocrine tumors. The operational steps are as follows: Figure 1 As shown, the specific steps include the following:

[0083] (1) Determine the types and concentrations of drugs in the drug sensitivity test and prepare a drug-containing culture medium of appropriate concentration;

[0084] (2) Observe and record the growth status of the PNET organoid model in the culture chamber of the drug sensitivity chip, and remove the original culture medium in the culture chamber;

[0085] (3) Add 45μL to 55μL of drug-containing culture medium to the two reservoirs on both sides of the drug sensitivity chip, and add 25μL to 35μL of drug-containing culture medium to the middle culture chamber of the drug sensitivity chip.

[0086] (4) Place the drug-sensitive chip after drug addition into the swing perfusion apparatus, set the conditions to 3° / 120min, and then place the swing perfusion apparatus into the incubator at 37°C and 5% CO2 for flow perfusion culture.

[0087] (5) On the third day, observe and record the growth status of the PNET organoid model and take bright-field images of the organoid using a high-content intelligent imaging analyzer.

[0088] (6) On day 5, observe and record the growth status of the PNET organoid model, take bright field photos of the organoid using a high-content intelligent imaging analyzer, and perform activity detection using a CTG detection kit and a chemiluminescent microplate reader.

[0089] (7) Based on bright-field cell images, comparative observations and analyses were conducted on organoid size and integrity between the control group and different drug-treated groups to clarify the growth inhibition and killing effects of different drugs on PNET organoids; cell viability and inhibition rate were calculated and analyzed based on cell viability test data. The cell viability calculation formula is: cell viability (%) = [A(drug-treated) - A(blank)] / [A(no-drug-treated) - A(blank)] × 100, inhibition rate (%) = 100 - cell viability (%).

[0090] The following examples illustrate the implementation of this application in more detail.

[0091] Example 1: Complex PNET organoid model constructed based on a 96-well plate

[0092] (1) 2D cell preparation: Take one cryopreservation tube each of vascular endothelial cells and cancer-related fibroblasts, and thaw and culture them separately. After the cells adhere to the wall on the second day, change the medium and continue to culture until the cells are completely adhered and grow and the confluence reaches 90%. Digest and centrifuge the cells to obtain cell pellet.

[0093] (2) PNET organoid preparation: Select one PNET organoid in culture at passage number P2 and continuously observe its status. It was observed that the organoid in this sample was in good condition and grew rapidly. After passage for 3-5 days, most organoids reached about 100μm.

[0094] (3) Collection of PNET organoids for later use: Collect the organoids being cultured according to the amount required by the drug sensitivity protocol. Collect a total of 10 wells, digest them until they are uniformly sized single cells or cell clusters of multiple cells, then stop digestion and set them aside for later use.

[0095] (4) Anti-adhesion treatment of 96-well U-shaped plate: According to the drug sensitivity test protocol, 63 wells of one U-shaped 96-well plate were treated. 100 μL of anti-adhesion reagent was added to each well, and the plate was left to stand in a clean bench for about 20 min. The anti-adhesion reagent was removed, and 100 μL of PBS was added to each well and left to stand for 10 min. The plate was washed twice.

[0096] (5) Preparation of cell suspension: The prepared PNET organoid cells, vascular endothelial cells and cancer-associated fibroblasts were mixed in a mixed culture medium at a cell number ratio of 1:0.5:0.4 to prepare a cell suspension;

[0097] (6) Plate preparation: Add 200 μL of cell suspension to each well of a 96-well U-shaped plate that has been treated with anti-adhesion. The total number of cells in each well is about 9,000. Place the 96-well plate that has been treated with anti-adhesion in a centrifuge and centrifuge at 400 g for 5 min.

[0098] (7) Incubation: The 96-well U-shaped plate after centrifugation was placed in a 37℃, 5% CO2 incubator and incubated for 2 days;

[0099] (8) Transferring cells: Take out the 96-well U-shaped plate, observe the cell spheroidization in the wells under a microscope, gently aspirate the culture medium in the wells, add 5 μL of matrix gel to each well, and gently and quickly transfer the complex model spheroids in 63 wells to the 96-well flat plate. Place the 96-well flat plate in a 37°C, 5% CO2 incubator and let the gel stand for 20 min to 30 min.

[0100] (9) Model construction and culture: Add 200 μL of mixed culture medium to each well, place the culture plate in an incubator at 37°C and 5% CO2 and let it stand to obtain a complex PNET organoid model.

[0101] The results of this embodiment are as follows: Figure 2 As shown, Figure 2 -A is a bright-field image (10X magnification) of the morphology of a cultured, ordinary, isolated PNET organoid. Figure 2 -B is a bright-field image (10X magnification) of the morphology of a complex PNET organoid model, created by... Figure 2 As can be seen, compared with ordinary standalone organoid models, the complex PNET organoid model prepared in this embodiment has a compact 3D tumor spheroid structure and is surrounded by outward-extending vascular structures, which more closely simulates the real structure of tumors and the tumor microenvironment.

[0102] Example 2: Drug susceptibility testing experiment based on a complex PNET organoid model using a 96-well plate.

[0103] (1) The model is constructed according to the construction method of the complex PNET organ model in Example 1;

[0104] (2) Drug preparation: First, the drug surufatinib was prepared as a high-concentration stock solution (10mM). On the day of drug administration, the stock solution was prepared as a 100nM working solution according to the experimental protocol, that is, the drug was mixed with the drug-free culture medium, and the volume of the working solution was 100*3*1.1μL;

[0105] (4) Adding drugs: After the constructed model is cultured for 1-2 days, until the blood vessels begin to extend outward, gently aspirate the culture medium in each well of the 96-well flat plate and add 100 μL of drug-containing culture medium to each well.

[0106] (5) Recording of PNET complex model morphology: Before drug administration (day 0): The appearance, size, and vascular growth of the PNET complex model were photographed and recorded using a high-content intelligent imaging analyzer. The appearance, size, and vascular characteristics of the model were also recorded at different time points after drug administration (days 1, 3, and 5) using the same high-content intelligent imaging analyzer.

[0107] (6) Activity assay: After taking photos on day 5, gently aspirate the culture supernatant, add 100 μL of CTG reagent mixing solution (CTG reagent: PBS = 1:1) to each well, shake for 5-10 min, and incubate at room temperature for 30 min. After the plate has finished incubating, use a chemiluminescent microplate reader for detection.

[0108] (7) Results Analysis: This includes morphological analysis and cell viability calculation and analysis. Cell morphological analysis includes observation and analysis of organoid size and integrity based on bright-field images of cells, clarifying the growth inhibition and killing effects of different drugs on complex PNET organoid models; the cell viability calculation formula is: Cell viability (%) = [A(drug-treated) - A(blank)] / [A(no-drug-treated) - A(blank)] × 100.

[0109] The experimental results of this embodiment are as follows: Figure 3 , Figure 4 As shown, by Figure 3 It can be seen that, compared with the control group (0 nM), surufatinib has a very significant killing effect on the PNET organoid complex model. 100 nM of the drug induced model rupture and angiogenesis apoptosis after one day of treatment. Activity was assessed on day 5 after treatment with 100 nM surufatinib. Figure 4 The calculated inhibition rates for the three duplicate wells and the average inhibition rate are shown.

[0110] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A composition for constructing complex PNET organoid models, characterized in that, It includes the following components: Based on the volume concentration of the final volume of the composition, Penicillin-streptomycin dual antibiotic solution, 1% vol / vol to 5% vol / vol; GlutaMax, 1%vol / vol~10%vol / vol; Serum-free supplements, wherein the serum-free supplements comprise 0.5% vol / vol to 10% vol / vol of B-27 and 1% vol / vol to 10% vol / vol of N-2; Wnt signaling pathway activator, 1%–20% vol / vol; Based on the final volume mass concentration of the composition, Human recombinant Noggin protein, 10 μg / L~500 μg / L; Human recombinant FGF10 protein, 50 ng / mL to 150 ng / mL; Based on the final volume molar concentration of the composition, HEPES, 1 mmol / L to 20 mmol / L; N-acetyl-L-cysteine, 0.2 μmol / L~5 μmol / L; Gastrin, 0.5 μmol / L~20 μmol / L; Nicotinamide, 0.1 mmol / L to 20 mmol / L; Adenylate cyclase activator, 2 μmol / L~20 μmol / L; ALK inhibitors, 0.1 μmol / L~10 μmol / L; ROCK inhibitors, 0.1 μmol / L~30 μmol / L; p38MAPK inhibitor, 0.05 μmol / L~10 μmol / L.

2. The composition according to claim 1, characterized in that, The Wnt signaling pathway activator includes one or more of R-spondin-1, BML-284, SM-04554, CHIR-99021, or HLY78; The adenylate cyclase activator is selected from one or both of Forskolin or NKH477; and / or, The ALK inhibitor is selected from one or more of A83-01, SB-505124, or TP-0184; and / or, The ROCK inhibitor is selected from one or both of Y27632 or HA-1077; and / or, The p38MAPK inhibitor is selected from one or more of SB202190, Adezmapimod, or Doramapimod.

3. The composition according to claim 1, characterized in that, The composition also includes a basal culture medium, which includes Advanced DMEM / F12 medium or DMEM medium.

4. A method for constructing a complex PNET organ-like model, characterized in that, The step includes culturing primary PNET organoids and / or passaged PNET organoids using the composition of any one of claims 1 to 3.

5. The construction method according to claim 4, characterized in that, The construction method specifically includes: S1: Pretreatment: Prepare cell pellets from vascular endothelial cells and fibroblasts for later use; digest primary or passaged PNET organoids to obtain uniformly sized cell clusters for later use; take a U-shaped bottom multi-well culture plate, add anti-adhesion reagent to each well, let stand, remove the anti-adhesion reagent, add PBS buffer to wash, let stand, and repeat washing 2-3 times. S2: Preparation of cell suspension: The composition is prepared into an organoid model culture medium, and then the organoid model culture medium is mixed with endothelial cell culture medium and fibroblast culture medium to prepare a mixed culture medium; the prepared PNET organoid cell clusters, vascular endothelial cell precipitate and fibroblast precipitate are mixed to prepare a mixed cell suspension; S3: Plating and culture. Add 3,000 to 15,000 cells to each well of the U-shaped bottom multi-well culture plate treated in S1, and then centrifuge. Inject mixed culture medium into the centrifuged U-shaped bottom multi-well culture plate and incubate for 1 to 2 days. S4: Rotate the ball, aspirate the culture medium in each well of the U-shaped bottom multi-well culture plate, add matrix gel, and transfer the complex PNET organoid model spheroids to the flat bottom multi-well culture plate; S5: Model construction and culture. Place a flat-bottomed multi-well culture plate in an incubator, let the gel stand still, add mixed culture medium, and place it in an incubator for static culture to obtain a complex PNET organoid model.

6. The construction method according to claim 5, characterized in that, In the mixed culture medium, the mass ratio of organoid model culture medium, endothelial cell culture medium and fibroblast culture medium is 1:(0.5-1):(0.1-0.5).

7. The construction method according to claim 5, characterized in that, The fibroblasts include cancer-associated fibroblasts; The cell number ratio of the PNET organoid cells, the cancer-associated fibroblasts, and the vascular endothelial cells is 1:(0.2-0.9):(0.1-0.8).

8. A complex PNET organ-like model, characterized in that, It is constructed using the construction method described in any one of claims 4 to 7.

9. The application of the complex PNET organoid model of claim 8 in the development of drugs for pancreatic neuroendocrine tumors.

10. The application according to claim 9, characterized in that, The applications include at least one of the following: drug screening, drug sensitivity testing, drug toxicity testing, or non-drug toxicity testing for pancreatic neuroendocrine tumors.

11. The application according to claim 10, characterized in that, The application includes at least the following steps: The test drug was added to the culture vector containing the complex PNET organoid model, and the culture was carried out for 3 to 5 days. The changes in the indicators of the complex PNET organoid model were then detected.

12. The application according to claim 11, characterized in that, The changes in the indicators include at least one of the following: changes in size, integrity, activity, and inhibition rate of the complex PNET organoid model.

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