Matrix cell hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and provides stromal cell hydrogel as well as a preparation method and application thereof. The stromal cell hydrogel is obtained by crosslinking stromal cells modified by azido groups N3, multi-arm polyethylene glycol and polyethylene glycol N3-PEG2K-N3 modified by diazido groups. According to the invention, the stromal cells are used as a gel component for tumor organoid or 3D culture for the first time. The living cells are used as a gel material, a tumor microenvironment is highly simulated, growth factors necessary for in-vitro culture of the tumor cells are provided, and the matrix cell hydrogel truly restores an in-vivo 3D culture environment. In addition, the matrix cell hydrogel can be used for patient PDX cell culture and can be used for large-scale drug screening.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a stromal cell hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] The main method for culturing tumor organoids or 3D cells is to inoculate tumor cells in a hydrogel, and the types of hydrogels mainly include Matrigel, alginate, gelatin, hyaluronic acid, agarose, laminin, collagen, fibrin and other gel components.

[0003] Stromal cells (TASC) include tumor endothelial cells (TECs), mesenchymal stem cells (MSCs), cancer-associated fibroblasts (CAFs), pericytes (PCs), and cancer-associated adipocytes (CAA). The tumor microenvironment consists of tumor cells, tumor stromal cells, and extracellular matrix. Stromal cells play an important role in the survival of hematological tumors.

[0004] 1. Role of Stromal Cells in the Tumor Microenvironment TASC, immune cells, and tumor cells together constitute the cellular components of the tumor microenvironment, and TASC accounts for approximately 50% of the total number of tumor tissue cells. Hematological tumors are highly dependent on the stromal microenvironment. CAFs promote tumor cell proliferation, invasion, metastasis, and drug resistance through multiple mechanisms. [1] CAFs secrete various cytokines including IL-1, IL-6, tumor necrosis factor, and chemokines, which constitute an important part of the cancer cell microenvironment and lead to drug resistance of cancer cells to various tumor treatment drugs. Stromal cells are key participants in regulating the immune response in the microenvironment of B-cell malignancies. Tumor cells and tissue-resident stromal cells, including endothelial cells (ECs), fibroblastic reticular cells (FRCs), and mesenchymal stromal cells (MSCs), have complex two-way interactions. Through cell-to-cell contact interactions, secretion of soluble factors, and extracellular vesicles (EVs), the activation of FRCs and MSCs is promoted, and these activations contribute to increased tumor survival and neovascularization. [1] In addition, ECs and FRCs in lymphoma upregulate immune checkpoints such as TIM-3 and PD-L1, and secrete immune regulatory factors such as IDO and IL-10, which block T cell proliferation and simultaneously activate immunosuppressive cells. [2] As the basis of lymphoma development, studies have shown that compared with non-tumor infiltrated lymph nodes, the gene expressions related to blood vessel development, accelerated angiogenesis, cell adhesion, and tumor metastasis in non-hematopoietic cells such as mesenchymal stem cells and endothelial cells in follicular lymphoma are significantly upregulated. [3]

[0005] 2. Related Research on Culturing Cells with Hydrogels Using human cells as a precursor of scaffold material for manufacturing living materials with similar tissue functions and cell programmability is a very promising method. Bioorthogonal reactions that integrate specific small chemical molecule groups into target biomolecules using the biosynthetic system of living life systems are generally divided into three types: metal-catalyzed bioorthogonal reactions, metal-free bioorthogonal reactions, and photocatalyzed bioorthogonal reactions.

[0006] In the past, co-culture models mainly involved co-inoculating stromal cells and tumor cells in Matrigel. Utilizing the characteristics of fibroblasts to migrate and adhere, they would grow in layers with tumor cells in Matrigel. In this invention, stromal cells are directly used as a gel component, enhancing the interaction between stromal cells and tumor cells. Summary of the Invention

[0007] The purpose of this invention is to provide a stromal cell hydrogel, its preparation method, and its application.

[0008] To achieve the purpose of this invention, this invention uses bioorthogonal click crosslinking to construct a stromal cell hydrogel for culturing hematological tumor cells.

[0009] In the first aspect, this invention provides a preparation method of a stromal cell hydrogel, which is obtained by crosslinking stromal cells modified with azide group N3 with multi-arm polyethylene glycol and polyethylene glycol modified with bis-azide group N3-PEG2K-N3.

[0010] Furthermore, the stromal cells modified with azide group N3 are obtained by treating stromal cells with N-azidoacetylmannosamine Ac4ManNAz, N-azidoacetylglucosamine Ac4GlcNAz, or N-azidoacetylgalactosamine.

[0011] In this invention, the stromal cells are mainly fibroblast cell lines, including but not limited to mouse or human fibroblast cell lines.

[0012] This invention uses living cell click chemistry to modify stromal cells: treating stromal cells with N-azidoacetylmannosamine Ac4ManNAz at a concentration of 5 - 200 μM (preferably 25 - 150 μM, more preferably 50 - 100 μM).

[0013] The multi-arm polyethylene glycol includes but not limited to DBCO-4Arm-PEG and / or DBCO-8Arm-PEG, preferably DBCO-4Arm-PEG.

[0014] Furthermore, the molar ratio of the reactive groups DBCO and N3 of DBCO-4Arm-PEG and N3-PEG2K-N3 participating in the reaction is between 1:0.2 and 1:2 (preferably between 1:0.2 and 1:0.5, more preferably 1:0.4).

[0015] In a specific embodiment of the present invention, the method for preparing the stromal cell hydrogel comprises the following steps: (1) Treat stromal cells with N-azidoacetylmannosamine Ac4ManNAz at a concentration of 50 - 100 μM to obtain stromal cells modified with azide group N3 at a concentration of 1.0E5 - 1.07E / 100 μl (preferably 3.0E5 - 1.06E / 100 μl, more preferably 0.5E6 / 100 μl); (2) Mix DBCO-4Arm-PEG with a mother liquor concentration of 10% and bis-azido polyethylene glycol with a concentration of 2% according to the molar ratio of the reactive groups DBCO to N3 of 1:0.2 - 1:2, supplement the volume to 50 μl with 10 mM Hepes buffer, mix well, and incubate at 37°C for 15 - 45 min. The final concentrations of DBCO-4Arm-PEG and bis-azido polyethylene glycol in the resulting mixed system are 5% - 9% and 0.2% - 1% respectively (preferably 5% and 0.4%); (3) Mix 30 - 80 μl of the stromal cells modified with azide group N3 in step (1) with 50 - 100 μl of the mixed system in step (2), supplement the volume to 130 μl with 10 mM Hepes buffer, and crosslink at 37°C for 15 - 45 min to obtain the product.

[0016] In a second aspect, the present invention provides a stromal cell hydrogel prepared according to the described method.

[0017] In a third aspect, the present invention provides the application of the described stromal cell hydrogel in the in vitro culture of hematological tumor cells.

[0018] In a fourth aspect, the present invention provides an anti-tumor drug screening model by mixing the described stromal cell hydrogel and hematological tumor cells in a quantity ratio of 1:5 - 1:20 (preferably 1:5 - 1:10) as the anti-tumor drug screening model.

[0019] In a fifth aspect, the present invention provides the application of the described model in the screening of anti-tumor drugs.

[0020] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The stromal cell gel of the present invention significantly prolongs the in vitro culture survival time of cells derived from tumor patients.

[0021] (2) The present invention for the first time uses stromal cells themselves as gel components for tumor organoids or 3D culture. Using living cells themselves as gel materials highly simulates the tumor microenvironment, provides growth factors necessary for the in vitro culture of tumor cells, and the stromal cell hydrogel truly restores the in vivo 3D culture environment.

[0022] (3) Glycosylation modification does not affect stromal cell behavior.

[0023] (4) PDX cell culture with stromal cell hydrogel can be used for large-scale drug screening. Description of the Drawings

[0024] Figure 1 This is the effect of Ac4ManNAz on the viability of various stromal cells detected by the CCK8 kit in the preferred embodiment of the present invention. Among them, A is the effect of Ac4ManNAz on the viability of MRC5 cells detected by the CCK8 kit. B is the effect of Ac4ManNAz on the viability of MRC5-LB cells detected by the CCK8 kit. C is the effect of Ac4ManNAz on the viability of C2C12 cells detected by the CCK8 kit. D is the effect of Ac4ManNAz on the viability of NIH3T3 cells detected by the CCK8 kit. E is the effect of Ac4ManNAz on the viability of MS5 cells detected by the CCK8 kit. F is the effect of Ac4ManNAz on the viability of HS5 cells detected by the CCK8 kit. G is the effect of Ac4ManNAz on the viability of MEF cells detected by the CCK8 kit.

[0025] Figure 2 This is to determine the cross-linking conditions of Ac4ManNAz with stromal cells in the preferred embodiment of the present invention. Among them, A is the confocal microscopy analysis of the cross-linking of Ac4ManNAz with stromal cells. Red is Cyanine5-DBCO staining, blue is Hoechst33342 staining, and the scale bar is 20 μm. B is the analysis of the fluorescence intensity change after cross-linking of Ac4ManNAz with stromal cells at different concentrations.

[0026] Figure 3 This is the optimal gel condition for constructing stromal cell hydrogel in the preferred embodiment of the present invention. Among them, 1:1:0 represents the molar ratio of the reactive groups DBCO and N3 of DBCO-4Arm-PEG and N3-PEG2K-N3 participating in the reaction in the gel and the amount of Ac4ManNAz-modified stromal cells added per 100 μl of the gel. 1:0.4:0.5E6, 1:0.4:1E6, and 1:0.4:2E6 respectively represent the amount of MRC5 cells treated with 50 μM Ac4ManNAz at 0.5E6 / 100 μl, 1E6 / 100 μl, and 1E6 / 100 μl based on the molar ratio of 1:0.4 of the reactive groups DBCO and N3 of DBCO-4Arm-PEG and N3-PEG2K-N3 participating in the reaction for 3 days.

[0027] Figure 4This is the gelation time point detected by the rheometer in the preferred embodiment of the present invention. On the basis of the molar ratio of 1:0.4 of the reactive groups of DBCO-4Arm-PEG and N3-PEG2K-N3, different amounts of MRC5 cells treated with 50 μM Ac4ManNAz were added, and the gel volume was 130 μl.

[0028] Figure 5 This is the hydrogel of MRC5 cells cultured for different times in the preferred embodiment of the present invention.

[0029] Figure 6 This is the change of live and dead cells of gel scaffolds detected by confocal microscopy in the preferred embodiment of the present invention at different culture times. Among them, A is the cell viability detection of MRC5 gel at 0 day, 3 days, 7 days, 14 days and 21 days. The green is MRC5 stained with CellTracker™ Green CMFDA, and the red is PI staining. B shows the viability of cells in MRC5-LB gel at 0 day, 3 days, 7 days, 14 days and 21 days. The green is MRC5-LB cells, the red is PI staining, and the scale bar is 50 μm.

[0030] Figure 7 This is the confocal microscopy detection result of MHG gel cultured for 7 days, 14 days and 21 days after CD20 antibody and ZombieViole staining. The green is MRC5 stained with CellTracker™ Green CMFDA, the cyan is HBL-1 stained with PE-cy7-anti-human CD20, and the red is dead cells stained with ZombieViole, and the scale bar is 50 μm.

[0031] Figure 8 This is the confocal microscopy analysis of MLPG gel cultured for different times in the preferred embodiment of the present invention. A is the MLPG gel cultured for 0 day, 1 day and 4 days. Among them, the green is MRC5-LB, and the red is PDX cells stained with CellTrace™ Far Red Cell Proliferation Kit, and the scale bar is 50 μm. B is the picture of MLPG gel cultured for 0 day, 7 days, 14 days and 21 days after CD20 antibody and ZombieViole staining. Among them, the green is MRC5-LB, the cyan is PDX cells stained with PE-cy7-anti-human CD20, and the red is dead cells stained with Zombie Viole, and the scale bar is 50 μm.

[0032] Figure 9 This is the killing efficiency of the stromal cell model treated with different drugs in the preferred embodiment of the present invention. A-C are the killing efficiencies of the MHG model, HG model and HBL-1 cells treated with different drugs respectively.

[0033] Figure 10 It shows the cell viability of other stromal cell hydrogel models in the present invention at different culture times. Among them, A shows the confocal detection results of ZombieViolet staining of other stromal cell hydrogel models in the present invention at 0 day and 3 days of culture. Green represents stromal cells stained with CellTracker™ Green CMFDA, red is OCI-Ly7 cells stained with CellTrace™ Far Red Cell Proliferation Kit, and purple represents dead cells stained with ZombieViolet. Scale bar: 50 μm. B shows the proportion of live cells of four stromal cell models at 0 day and 3 days of culture. Detailed implementation manners

[0034] The present invention provides a stromal cell hydrogel culture system for a new method of culturing hematological tumor organoids. The stromal cell hydrogel culture system of the present invention is used to solve the problems of short in vitro culture time and difficult survival of hematological tumor cells, and at the same time provides a 3D tumor model that can simulate the tumor microenvironment for large-scale screening of clinical drugs for hematological tumors.

[0035] The present invention adopts the following technical solutions: The present invention provides a method for constructing a stromal cell hydrogel based on bioorthogonal reaction to culture lymphoma organoids.

[0036] The present invention provides a stromal cell gel. Fibroblasts modified with azide group N3 are crosslinked with DBCO polyethylene glycol (multi-arm polyethylene glycol) and bis-azide polyethylene glycol to obtain a stromal cell hydrogel.

[0037] The stromal cells involved in the present invention are mainly fibroblast cell lines, including but not limited to mouse or human fibroblast cell lines.

[0038] Mouse fibroblast cell lines, such as: mouse embryonic fibroblasts (NIH / 3T3), mouse fibroblasts (L929), mouse embryonic fibroblasts (3T3-L1), mouse embryonic fibroblasts (C3H 10T1 / 2), mouse embryonic fibroblasts (BALB / 3T3 clone A31), CF-1 mouse embryonic fibroblasts (CF-1 MEF), mouse fibroblasts (PA317), mouse embryonic fibroblasts (STO), mouse embryonic fibroblasts (Psi2 DAP), rat cardiac fibroblasts (RCF), mouse lung fibroblasts (Mlg 2908), mouse embryonic fibroblasts (SNL76 / 7), mouse embryonic fibroblasts (Psi2DAP), mouse fibroblasts (NCTC clone 929), mouse fibroblasts (McCoy), mouse lung fibroblasts (WML2), mouse cardiac fibroblasts (MCFs), mouse myoblasts (C2C12), mouse embryonic fibroblasts (MEF), mouse bone marrow stromal cells (MS-5), etc.

[0039] Human fibroblast cell lines: human skin fibroblasts (HFF-1), human embryonic lung fibroblasts (HFL-I), human embryonic lung fibroblasts (HEL), human skin fibroblasts (HDF-α), human gingival fibroblasts (HGF-1), human embryonic lung fibroblasts (IMR-90), human skin fibroblasts (BJ), human skin fibroblasts (HSF), human embryonic lung fibroblasts (MRC5), human embryonic skin fibroblasts (CCC-ESF-1), human periodontal ligament fibroblasts (HPDLF), human embryonic lung fibroblasts (CCC-HPF-1), human pulmonary artery fibroblasts (STR), human lung fibroblasts (HPF), human dermal fibroblasts (HDF), human bronchial fibroblasts (HBF), human prostate fibroblasts (HPrF), human uterine fibroblasts (HUF), human ovarian fibroblasts (HOF), human renal fibroblasts (HRF), human bladder fibroblasts (HBdSF), human cardiac fibroblasts (HCFB), human brain fibroblasts (HBVAF), human fibroblast-like synoviocytes (HFLS), human embryonic lung fibroblasts (IMR-90), human foreskin fibroblasts (HFF-1), human skin fibroblasts (GM0637), human periodontal ligament fibroblasts (HPDLF), human connective tissue fibroblast-like cells (L Cells), human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA), etc., tumor-associated fibroblasts.

[0040] Metabolic glycoprotein labeling reagents for modifying stromal cells using live cell click chemistry mainly include tetra-acylated N-azidoacetyl mannosamine Ac4ManNAz, N-azidoacetyl glucosamine Ac4GlcNAz, and N-azidoacetyl galactosamine Ac4GalNAz. The labeled azide N3 stromal cells are crosslinked with multi-arm polyethylene glycol (DBCO-4Arm-PEG, DBCO-8Arm-PEG) and a small amount of bis-azido polyethylene glycol (N3-SS-PEG-SS-N3, N3-PEG-N3, N3-PEG-SS-N3, N3-PEG2K-SH, N3-PEG-SS-PEG-N3) and a polypeptide chain modified with azidoacetic acid (azidoacetic acid - "X1""Y""X2"-K(N3)) to obtain a stromal cell hydrogel. Among them, the molecular weight of the small amount of bis-azido polyethylene glycol (N3-SS-PEG-SS-N3, N3-PEG-N3, N3-PEG-SS-N3, N3-PEG-SH, N3-PEG-SS-PEG-N3) is preferably 400 - 2000 g / mol, and most preferably 2000 g / mol. The relative molecular mass of azidoacetic acid - "X1""Y""X2"-K(N3) is 800 - 3000 g / mol; X = 1 - 5 amino acids, and the amino acids are G or R; Y is VPMS / MRGG, IPVS / LRSG, SGESPAY / YTA, RPFS / MIMG, VPLS / LTMG, VPLS / LYSG, GGYAE / LRMGG, GPLG / LWAR, IPES / LRAG, and GGPLG / LYAGG, etc.

[0041] The present invention also provides a method for using stromal cell hydrogels in 3D culture of hematological malignancies. It includes: hematological malignancy cell lines, lymphoma cell lines (HBL-1, OCI-Ly7), leukemia cell lines, multiple myeloma cell lines and other hematological system tumor cell lines. Lymphoma PDX cells and patient-derived primary lymphoma cells. Collect hematological malignancy cells and add them to the mixture of stromal cells MRC5 treated with N3-PEG2K-N3 and Ac4ManNAz or MRC5 (MRC5-LB) cells overexpressing CD40L and BAFF. After thorough mixing, further mix well with DBCO-4Arm-PEG. Transfer the hydrogel of stromal cells containing hematological malignancies to a confocal dish, incubate at 37 °C for 30 min, then add the culture medium, and culture in an incubator at 37 °C with 5% CO2. The use concentration of Ac4ManNAz is preferably 5 - 200 μM, more preferably 50 - 150 μM, and most preferably 50 - 100 μM. The molar ratio of DBCO-4Arm-PEG to the groups participating in the reaction of N3-PEG2K-N3 is preferably between 1:0.2 and 1:2, more preferably between 1:0.2 and 1:0.5, and most preferably 1:0.4. The concentration of stromal cells treated with Ac4ManNAz is preferably 1.0E5 - 1.07E / 100 μl, more preferably 3.0E5 - 1.06E / 100 μl, and most preferably 0.5E6 / 100 μl. The ratio of the number of stromal cells to hematological malignancy cells or PDX cells is preferably 1:5 - 1:20, more preferably 1:5 - 1:10. At different times, perform viability identification and laser confocal scanning microscopy analysis on the stromal cell hydrogels culturing hematological malignancy cells.

[0042] The present invention also provides a model for in vitro drug sensitivity detection. This model is a stromal cell hydrogel of cells derived from hematological malignancy patients.

[0043] Cells derived from patients are PDX tissue blocks or patient surgical puncture samples, etc. Hematological malignancies include B-cell lymphoproliferative diseases and tumors and T- and NK-cell lymphoproliferative diseases and tumors. Each subtype includes diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, peripheral T-cell lymphoma, myeloproliferative neoplasms, anaplastic large cell lymphoma, and multiple myeloma, etc.

[0044] The test drugs include cyclophosphamide, doxorubicin, dexamethasone, vincristine, cytarabine, carboplatin, cisplatin, oxaliplatin, gemcitabine, ifosfamide, etoposide, prednisone, bendamustine, lenalidomide, ibrutinib, zanubrutinib, acalabrutinib, pirtobrutinib, chidamide, tazemetostat, rituximab, obinutuzumab, acuretozumab, gefitinib (Gao Luo Hua), motuzumab, polatuzumab (You Luo Hua), tenesitumab, etc. For different drugs, three concentrations of high, medium and low were designed to detect the sensitivity of the stromal cell hydrogel tumor model to the drugs, and then the viability of tumor cells in the hydrogel was detected by cell viability detection reagents such as CCK-8 or MTT. The method for identifying cell death and survival was that the stromal cell hydrogels containing tumor cells from patients with hematopoietic and lymphoid tissue tumors cultured for different times were stained with Hoechst33342 and live-dead cell staining reagents and then analyzed by a high-content cell imaging system.

[0045] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0046] Ac4ManNAz and Cyanine5-DBCO used in the following examples were both purchased from MedChemExpress, and N3-PEG2K-N3 and DBCO-4Arm-PEG were both purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0047] Human embryonic lung fibroblasts (MRC5-LB) overexpressing CD40L and BAFF were constructed by our laboratory through lentiviral transfection. The specific construction process is as follows: 24 h after plating 5E4 - 1E5 MRC5 cells in a well plate, calculate the volume of lentivirus to be added according to the number of added cells, where the lentivirus MOI is between 10 - 40. After transfection for 16 - 20 h, discard the culture medium supernatant, add a culture medium containing 0.1 mg / mL puromycin for screening for 3 days, collect the cells, and detect the transfection efficiency by flow cytometry.

[0048] Example 1 Preparation of stromal cell hydrogel This example provides a preparation method using stromal cells as a hydrogel scaffold.

[0049] The raw materials for the preparation of the stromal cell hydrogel in this embodiment include: (1) Preparation of the medium for human embryonic lung fibroblasts MRC5-LB overexpressing CD40L and BAFF: Add 10% Gibco serum, 100 U / ml penicillin, and 100 U / ml streptomycin to DMEM high-glucose medium. (2) Preparation of the medium for human embryonic lung fibroblasts MRC5: Add 10% BI serum, 100 U / ml penicillin, and 100 U / ml streptomycin to DMEM high-glucose medium. (3) Different concentrations of Ac4ManNAz. (4) CCK8 kit. (5) Cyanine5-DBCO. (6) N3-PEG2K-N3. (7) DBCO-4Arm-PEG.

[0050] This embodiment provides a preparation method using stromal cells as a hydrogel scaffold, and the specific steps are as follows: (1) Identification of the effect of Ac4ManNAz on cell viability Seed MRC5 cells or MRC5-LB cells into a 96-well plate at a density of 3E3 per well. After culturing at 37 °C for 24 h, discard the medium and add DMEM complete medium containing different concentrations of Ac4ManNAz. The concentrations of Ac4ManNAz include 0 μM, 5 μM, 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, and 200 μM. After culturing at 37 °C for 3 days, discard the medium, add 100 μL of fresh DMEM complete medium, and add 10 μl of CCK8 solution to each well. Incubate at 37 °C for 4 hours. Detect the absorbance at 450 nm using an enzyme-labeled instrument. The experimental results are shown in Figure 1 . Figure 1 A shows the effect of different concentrations of Ac4ManNAz on the viability of MRC5 cells. Figure 1 B shows the effect of different concentrations of Ac4ManNAz on the viability of MRC5-LB cells. As Figure 1 shown in A-D, there is no significant difference in the viability of MRC5, MRC5-LB, C2C12, and NIH3T3 cells at Ac4ManNAz concentrations of 0-100 μM. As Figure 1 shown in E-G, there is no significant change in the viability of MS5, HS5, and MEF cells at Ac4ManNAz concentrations of 0-50 μM.

[0051] (2) Determination of Ac4ManNAz cross-linked stromal cells The MRC5 cells were seeded into confocal dishes, and different concentrations of Ac4ManNAz were added, followed by culturing at 37 °C for 3 days. The MRC5 cells were washed twice with DMEM basal medium. Cyanine5-DBCO with a final concentration of 2.5 μM was added to 1 ml of DMEM for reaction, and the cells treated without Ac4ManNAz were used as the blank control. Incubate at 37 °C for 15 min. After incubation, the medium was discarded, and the cells were washed with 1 ml of DMEM medium. Then, DMEM containing Hoechst33342 was added for staining, with a final concentration of Hoechst33342 of 20 μM. Incubate at room temperature for 20 min, and then the staining results were photographed under a confocal microscope. The experimental results are shown in Figure 2 . As Figure 2 shown in A, when the concentration was between 5 μM and 50 μM, the fluorescence intensity gradually increased with the increase in the concentration of Ac4ManNAz. There was no significant difference in the fluorescence intensity between 50 μM, 75 μM, and 100 μM, and the fluorescence intensity was significantly stronger than that of the untreated group, indicating that a large amount of Ac4ManNAz was conjugated to the stromal cells. Figure 2 B shows that the average fluorescence intensity increased significantly with the increase in the concentration of Ac4ManNAz.

[0052] (3) Explore the optimal conditions for constructing stromal cell hydrogels To explore the optimal conditions for constructing stromal cell hydrogels, the mother liquor concentrations of 10% DBCO-4Arm-PEG and 2% N3-PEG2K-N3 were mixed at the molar ratios of 1:1, 1:0.4, and 1:0.2 of the reactive groups DBCO and N3 involved in the reaction. 10 mM Hepes was added to make up the volume to 50 μl. After mixing, incubate at 37 °C for 30 min. The final concentrations of DBCO-4Arm-PEG and N3-PEG2K-N3 in the resulting mixed system were 5% and 1%, 5% and 0.4%, 5% and 0.2% respectively. The results are shown in Figure 3 . As Figure 3 shown, when the molar ratio of the reactive groups DBCO and N3 of DBCO-4Arm-PEG and N3-PEG2K-N3 involved in the reaction was 1:0.4, a gel could not be formed. However, MRC5 cells treated with 0.5E6 / 100 μl, 1E6 / 100 μl, and 2E6 / 100 μl of Ac4ManNAz respectively could form a gel at this molar ratio. Therefore, the subsequent optimal conditions for constructing stromal cell hydrogels were selected as the system with mother liquor concentrations of 10% DBCO-4Arm-PEG and 2% N3-PEG2K-N3 and a molar ratio of 1:0.4 of the reactive groups DBCO and N3 involved in the reaction.

[0053] Under the above conditions, MRC5 cells treated with 0.5E6 / 100 μl, 1E6 / 100 μl, and 2E6 / 100 μl of Ac4ManNAz were added respectively, and the volume was supplemented to 130 μl with 10 mM Hepes (that is, 39 μl of MRC5 cells treated with Ac4ManNAz were mixed with 91 μl of the above mixed system, and the volume was supplemented to 130 μl with 10 mM Hepes). The rheometer panel was preheated at 37 °C for 2 min. After the sample was mixed evenly, it was immediately transferred onto the rheometer panel, and the stromal cell hydrogel material was characterized by the rheometer. The results are shown in Figure 4 . As Figure 4 shown, a gel was formed 140 s after adding MRC5 cells treated with 0.5E6 / 100 μl of Ac4ManNAz. Gels were rapidly formed after adding 1E6 / 100 μl and 2E6 / 100 μl of cells respectively. Therefore, MRC5 stromal cells treated with 0.5E6 / 100 μl of Ac4ManNAz were selected to construct the stromal cell hydrogel.

[0054] Example 2 Detection of the stability of the stromal cell hydrogel In this example, the stability of the stromal cell hydrogel was detected.

[0055] (1) Stability of the stromal cell hydrogel cultured for different times The MRC5 stromal cell hydrogel was prepared by the above method, with a gel volume of 40 μl, and cultured in DMEM complete medium for different times. Its stability was detected, and the photographed results are shown in Figure 5 . As Figure 5 shown, compared with Day0, the volume of the gels cultured for different times increased, and there was no obvious difference in the gel state.

[0056] (2) Detection of the viability of stromal cells in the gel The MRC5 hydrogel was prepared, and the stromal cells treated with Ac4ManNAz were stained with CellTracker™ Green CMFDA. It was cultured in a 48-well plate, and 300 μl of DMEM complete medium was added and cultured at 37 °C for different times. The viability of MRC5 cells in the MRC5 cell hydrogel cultured for different times was determined by the live and dead cell staining method. The MRC5 hydrogel at different culture time points was taken, the DMEM complete medium in the well plate was aspirated, washed once with PBS, PBS containing PI was added, stained at room temperature for 15 min, and the staining results were detected by confocal microscopy. As Figure 6 shown in A, there were some dead cells in the MRC5 hydrogel in the first 3 days of culture. However, as the culture time extended, the number of dead cells in the MRC5 hydrogel decreased, and a large number of live MRC5 cells still existed in the hydrogel at 21 days of culture, and dead cells were extremely rare.Figure 6 As shown in B, although there are some dead cells in the MRC5-LB hydrogel in the first 3 days of pre-culture, with the extension of the culture time, the viability of MRC5-LB in the gel gradually increases. By day 21, a large number of MRC5-LB can still survive. This indicates that the gel formed by DBCO-4Arm-PEG and N3-PEG2K-N3 does not affect the growth of the above stromal cells within 21 days.

[0057] Example 3: Culturing cells with stromal cell hydrogel This example provides a method for culturing tumor cells with stromal cell hydrogel.

[0058] The raw materials for culturing tumor cells with stromal cell hydrogel in this example include: (1) The medium OCM1 for culturing PDX cells: DMEM high-glucose medium added with 10% Gibco serum, 100 U / ml penicillin and 100 U / ml streptomycin.

[0059] (1) Culturing HBL-1 with MRC5 hydrogel Prepare the MRC5 hydrogel (MHG) for culturing HBL-1 by the above method. The stromal cells treated with Ac4ManNAz are stained with CellTracker™ Green CMFDA and then mixed with HBL-1 to prepare MHG. The gel volume is 10 μl, and the ratio of the number of MRC5 cells to HBL-1 cells is between 1:5 and 1:20. The MHG hydrogel is cultured in the complete DMEM medium dedicated to MRC5 for different times. Take the MHG cultured for different times, discard the medium, wash it once with PBS, add a solution containing Anti-human-CD20 and Zombie Violet dyes, stain at 37 °C for 15 min, and then take pictures of the stained results under a confocal microscope. The results are shown in Figure 7 . As shown in the figure, HBL-1 proliferates significantly between Day0 and Day7, and the cell state is good. However, on Day14, the number of apoptotic cells of HBL-1 increases. This indicates that under the condition of this stromal cell gel, HBL-1 can grow stably for at least 7 days.

[0060] (2) Culturing PDX cells with MRC5-LB hydrogel Obtain PDX cells. Rapidly dissolve the cryopreserved tissue blocks at 37 °C, transfer all the tissue to 6 ml of buffer, and rinse the cryotube. Centrifuge at 1200 rpm for 5 min, discard the supernatant, add 2 mL of buffer to resuspend, blow and suck thoroughly with a dropper, after standing for 20 s, transfer the supernatant to a filter. Add 1 ml of buffer to resuspend the tissue, transfer all the samples to a 1.5 ml centrifuge tube with a dropper, after standing, aspirate the supernatant and filter it through a 70-mesh sieve into a 6 cm dish. Add 500 μl of buffer to the 1.5 ml centrifuge tube, cut the PDX into pieces with scissors, then add 500 μl of buffer and mix well, and then transfer all to a 5 ml centrifuge tube. Add 1 ml of buffer and mix well, stand for about 20 s, collect the cells in the supernatant, filter through a 70-mesh sieve, and aspirate the filtered cells and transfer them to a 15 ml centrifuge tube. Wash twice with buffer, and at the same time repeat the above physical operation to cut the PDX tissue until the supernatant in the final 5 ml centrifuge tube is clear. Transfer all the cells in the collected supernatant to a filter, aspirate the filtered cells and transfer them to a 15 ml centrifuge tube, take 20 μl of the mixed cell suspension for counting, and the obtained cells are PDX cells.

[0061] Prepare MRC5-LB hydrogel (MLPG) for culturing PDX cells, where the ratio of MRC5-LB to the number of PDX cells is between 1:5 and 1:20. Stain the PDX cells with the CellTrace™ Far Red Cell Proliferation Kit for 20 min, and then culture them with MRC5-LB gel. At 0 day, 1 day, and 4 days of culture, confocal detection was used to detect the changes of cells in MLPG, and the results are shown in Figure 8 A. Another prepared MLPG was cultured in a 96-well plate, 300 μl of OCM1 medium was added, and cultured at 37 °C for different days. Take the MLPG cultured for different times, discard the medium, wash twice with PBS, add Anti-human-CD20 and Zombie Violet dyes, stain at room temperature for 15 min, and then take pictures of the stained results under a confocal microscope. The results are shown in Figure 8 B. As Figure 8 Shown in B, on the 14th day of culture, PDX cells were adsorbed on MRC5-LB cells. Although some PDX cells died, most PDX cells were in good condition.

[0062] In this embodiment, the preparation method of MRC5 hydrogel (MHG) is as follows: (1) Treat MRC5 cells with Ac4ManNAz at a concentration of 50 μM to obtain Ac4ManNAz-treated MRC5 cells at 0.5E6 / 100 μl; (2) Mix DBCO-4Arm-PEG with a mother liquor concentration of 10% and N3-PEG2K-N3 with a concentration of 2% at a molar ratio of DBCO to N3 of 1:0.4 for the groups participating in the reaction, and supplement the volume to 50 μl with 10 mM Hepes buffer. After mixing, incubate at 37 °C for 30 min. The concentrations of DBCO-4Arm-PEG and N3-PEG2K-N3 in the resulting mixed system are 5% and 0.4% respectively. (3) Mix 30 μl of the Ac4ManNAz-treated MRC5 cells from step (1) with 91 μl of the mixed system from step (2), supplement the volume to 130 μl with 10 mM Hepes buffer, and crosslink at 37 °C for 30 min to obtain the product.

[0063] The preparation method of MRC5-LB hydrogel (MLPG) is the same as that of MRC5 hydrogel (MHG).

[0064] Example 4: Using stromal cell hydrogel for drug screening This example provides a method for screening tumor drugs using stromal cell hydrogel.

[0065] Prepare the MHG hydrogel by the above method, with the gel volume being 1 - 10 μl, preferably 1 - 5 μl, and more preferably 1 - 2 μl. The ratio of the number of MRC5 cells to HBL-1 cells is between 1:5 and 1:20. At the same time, use the gel without MRC5 cells (HG) as a control. In this group, the molar ratio of the reactive groups DBCO and N3 of DBCO-4Arm-PEG and N3-PEG2K-N3 participating in the reaction is 1:1, and the number of added HBL-1 cells is the same as that in the MHG gel. Additionally, use the group of HBL-1 cultured alone without gel as a positive control. After culturing the MHG and HG hydrogels for 1 - 3 days, add different concentrations of Ibrutnib, SHR2554, and Doxorubicin respectively. In the HBL-1 group, add different concentrations of Ibrutnib and SHR2554 (where Ibrutnib is a BTK inhibitor, a new targeted anti-cancer drug for the treatment of various cell lymphomas; SHR2554 is an EZH2 inhibitor used for the treatment of EZH2 abnormalities, lymphoma, and multiple myeloma; Doxorubicin is an anthracycline broad-spectrum antibiotic). Culture for 1 - 3 days, with the final concentration of Ibrutnib being 1 μM and 10 μM, the final concentration of SHR2554 being 12.5 μM and 25 μM, and the final concentration of Doxorubicin being 5 μM and 25 μM. Discard the culture medium, wash twice with PBS, add dyes containing DAPI and PI, stain at 37°C for 30 min, and then detect the experimental results on a high-content cell imaging and analysis system. The HBL-1 group uses the CellTiter-Glo® Luminescent Cell Viability Assay reagent to detect the cell viability after drug treatment. The results are shown in Figure 9 . As Figure 9 shown, MHG is the most sensitive to Doxorubicin. Under the same culture conditions, the proportion of dead cells in HBL-1 increases with the increase in drug concentration. After treatment with the same concentration of Ibrutnib and SHR2554 in the MRC5 / HBL-1 Gels and HBL-1 Gel groups, the killing effect of the drug on HBL-1 in the HBL-1 Gel group is significantly higher than that in the MRC5 / HBL-1 Gels group, and the killing efficiency of both groups is significantly lower than that in the HBL-1 group. This shows that HBL-1 is sensitive to the drugs, the sensitivity of HBL-1 under 3D culture (HBL-1 Gel group) to the drugs decreases, and the cell killing in the stromal cell gel group (MRC / HBL-1) is the smallest. The presence of stromal cells reduces the sensitivity of HBL-1 to the above drugs.

[0066] Example 5 Other potentially applicable stromal cell hydrogels for drug screening This example provides other potentially applicable stromal cell hydrogels for tumor drug screening.

[0067] Prepare the stromal cell hydrogels for culturing OCI-Ly7 by the above method, including C2C12 culturing OCI-ly7 (C2OCIG), NIH3T3 culturing OCI-Ly7 (NIOCIG), MEF culturing OCI-Ly7 (MEOCIG), and MS5 culturing OCI-Ly7 (MSOCIG). The OCI-Ly7 cells were stained with the CellTrace™ Far Red Cell Proliferation Kit for 20 min, and the above stromal cells treated with 50 μM Ac4ManNAz for 3 days were stained with CellTracker™ Green CMFDA respectively to prepare the stromal cell hydrogels for culturing OCI-Ly7. The gel volume was 10 μl, and the ratio of the number of stromal cells to OCI-Ly7 cells was between 1:5 and 1:20. The stromal cell hydrogels were cultured in OCM1 for 3 days. Take the stromal cell hydrogels on Day0 and Day3 of the culture, discard the culture medium, wash twice with 10 mM Hepes respectively, then add 10 mM Hepes containing Zombie Violet dye, and take pictures of the staining results under a confocal microscope after staining at 37 °C for 15 min. The results are shown in Figure 10 . As Figure 10 shown in A, on Day3 of the culture, due to the excessive proliferation of OCI-Ly7 cultured by NIH3T3, the viability of the cells decreased significantly on Day3, which may be caused by limited space and insufficient nutrients. This phenomenon can be avoided by reducing the ratio of stromal cells to tumor cells on the day of preparation. Although some of the OCI-Ly7 cells cultured by C2C12 died, through counting, as Figure 10 shown in B, the viability of OCI-Ly7 cultured by the three stromal cells of C2C12, MEF, and MS5 was all above 80% on Day3, indicating that these three stromal cell hydrogels can be used for drug screening.

[0068] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

[0069] References: [1] de Visser K E, Joyce J A. The evolving tumor microenvironment:From cancer initiation to metastatic outgrowth[J]. Cancer Cell. 2023 Mar 13;41(3):374-403. [2] Turley S J. Immunological hallmarks of stromal cells in the tumour microenvironment[J]. Nat Rev Immunol. 2015.15(11):669-82. [3] Abe Y. A single-cell atlas of non-haematopoietic cells in human lymph nodes and lymphoma reveals a landscape of stromal remodelling[J]. Nat Cell Biol. 2022 Apr.24(4):565-578.

Claims

1. A method for preparing a matrix cell hydrogel, characterized in that: The matrix cells modified with the azide group N3 are cross-linked with multi-arm polyethylene glycol and bis-azide modified polyethylene glycol N3-PEG2K-N3 to obtain; Wherein, the stromal cells modified with the azide group N3 are obtained by treating stromal cells with N-azidoacetylmannosamine Ac4ManNAz, N-azidoacetylglucosamine Ac4GlcNAz or N-azidoacetylgalactosamine; The stromal cells are of fibroblastic cell line.

2. The method according to claim 1, characterized in that Stromal cells were treated with N-azidoacetylmannosamine Ac4ManNAz at concentrations ranging from 5 to 200 μM.

3. The method according to claim 1, characterized in that The multi-arm polyethylene glycol is DBCO-4Arm-PEG and / or DBCO-8Arm-PEG.

4. The method according to claim 3, characterized in that The molar ratio of the groups DBCO and N3 involved in the reaction in DBCO-4Arm-PEG and N3-PEG2K-N3 is between 1:0.2 and 1:

2.

5. The method according to claim 4, characterized in that The following steps are involved: (1) Treating stromal cells with N-azidoacetylmannosamine Ac4ManNAz at a concentration of 50-100 μM to obtain stromal cells modified with azide groups N3 at a concentration of 1.0E5-1.07E / 100 μl; (2) Mix the mother solutions containing 10% DBCO-4Arm-PEG and 2% N3-PEG2K-N3 at a molar ratio of 1:0.2-1:2 between the groups DBCO and N3 involved in the reaction, add 10 mM Hepes buffer to make up the volume to 50 μl, mix well, and incubate at 37°C for 15-45 min. The final concentrations of DBCO-4Arm-PEG and N3-PEG2K-N3 in the resulting mixture are 5%-9% and 0.2%-1%, respectively. (3) Mix 30-80 μl of the matrix cells modified with the azide group N3 in step (1) with 50-100 μl of the mixed system in step (2), add 10 mM Hepes buffer to make up the volume to 130 μl, and cross-link at 37°C for 15-45 min.

6. The method according to any one of claims 1 to 5, characterized in that The fibroblast cell lines are derived from mice or humans.

7. A matrix cell hydrogel prepared according to the method of any one of claims 1 to 6.

8. Use of the matrix cell hydrogel according to claim 7 in the in vitro culture of blood tumor cells.

9. An anti-tumor drug screening model, characterized in that: The matrix cell hydrogel according to claim 7 is mixed with blood tumor cells in a ratio of 1:5-1:20 to serve as an anti-tumor drug screening model.

10. Use of the model according to claim 9 in screening anti-tumor drugs.

Images