Lymphoma organ as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a lymphoma organ as well as a preparation method and application thereof. The method comprises the step of performing in-vitro co-culture on lymphoma cells and engineered stromal cells or patient-derived tumor-associated fibroblasts. By co-culturing with engineered stromal cells or patient-derived tumor-associated fibroblasts, B-cell lymphoma cells can stably survive in vitro in a three-dimensional structure having multicellular components. The lymphoma organoid obtained according to the method is used as an anti-tumor drug screening model, and the model not only can better simulate the growth state of B cell lymphoma in vivo, but also can provide a research platform for individualized drug sensitivity and large-scale drug screening of B cell lymphoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a lymphoma organoid and a preparation method and application thereof. BACKGROUND

[0002] Lymphoma mainly includes Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL), and is a group of heterogeneous lymphoid tissue proliferative diseases. B-cell lymphoma (BCL) accounts for almost 95% of all lymphoma cases [1] . Although the treatment of B-cell lymphoma has entered the era of targeted chemotherapy, 40% of patients cannot be cured after first-line therapy (R-CHOP) and develop into relapsed / refractory B-cell lymphoma. B-cell lymphoma is highly heterogeneous, with many subtypes, and personalized precision treatment based on molecular typing is the trend [2] .

[0003] Lymphoma is a cancer that is highly dependent on tumor microenvironment. Currently, lymphoma cells derived from patients are difficult to maintain long-term survival in vitro, and the support of stromal cells is crucial for the survival of lymphoma cells in vitro. Stromal cells provide support for the growth and migration of B-cell lymphoma cells and secrete growth factors to promote the survival of lymphoma cells. CD40 ligand (CD40L) and B-cell activating factor (BAFF) play an important role in the survival of normal B cells and B-cell lymphoma [3] . Follicular helper T cells secrete CD40L and interleukin 4 (IL-4) to support the growth of follicular lymphoma cells, and the secretion of BAFF is also crucial for the survival of diffuse large B-cell lymphoma (DLBCL) cells. CD40L is mainly expressed by helper T cells, and BAFF is mainly expressed in myeloid innate immune cells. Studies on B-cell signaling pathways have shown that both CD40L and BAFF regulate the function of B cells by stimulating the activation of NF-кB, MAPKs and PI3K-AKT signaling pathways. IL-4 stimulates the proliferation of activated B cells and T cells, differentiates B cells into plasma cells, induces B cells to switch to IgE, and up-regulates the expression level of MHC class II antigens.

[0004] With the development of tumor organoid technology, tumor organoids are increasingly used in individualized drug efficacy evaluation of tumors, and are an ideal model for precision treatment [4] . However, the current tumor organoid technology is mainly suitable for the culture of epithelial tumors, and the culture conditions of lymphoma and the classic WNER (Wnt-3a / Noggin / EGF / RSPO1) organoid culture scheme for epithelial tumors are quite different, so the existing organoid culture technology is not suitable for the establishment of lymphoma organoids[5,6] Therefore, it is urgent to develop a method for constructing B-cell lymphoma organoids to provide a more accurate in vitro research model for personalized treatment of lymphoma and a new tool for new drug development and mechanism research.

[0005] New drug development and individualized drug efficacy evaluation of lymphoma depend on the development of preclinical disease models of lymphoma. Current disease models of B-cell lymphoma include tumor cell lines and patient-derived xenograft (PDX) models [7] Tumor cell lines grow rapidly and are relatively low-cost, and have made indelible contributions to the study of lymphoma pathogenesis and drug development, but cell lines lack tumor microenvironment and are difficult to reproduce the real situation in vivo in response to drugs; PDX models have an in vivo environment that can better simulate tumor biological characteristics, but the construction cycle is longer and the success rate is lower, limiting their large-scale use. SUMMARY

[0006] The purpose of the present application is to provide a lymphoma organoid and a preparation method and application thereof.

[0007] To achieve the purpose of the present application, in a first aspect, the present application provides a culture method of lymphoma organoids PDO, which comprises: co-culturing lymphoma cells with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF in vitro.

[0008] In the present application, the lymphoma cells are B-cell lymphoma cells from patient-derived xenograft cells PDXC or PDC.

[0009] The engineered stromal cells ESC are fibroblasts or stromal cells overexpressing CD40L and BAFF and / or other cytokines promoting lymphoma growth. Further, the fibroblasts or stromal cells can be derived from mice or humans, such as human fetal lung fibroblasts (MRC5), human bone marrow stromal cells (HS-5), mouse bone marrow stromal cells (MS-5), etc.

[0010] Preferably, the construction method of the engineered stromal cells ESC comprises: constructing a lentiviral vector, stably transfecting CD40L and BAFF into fibroblasts (such as human fetal lung fibroblasts MRC5) to obtain engineered stromal cells ESC.

[0011] Further, the preparation method of the patient-derived tumor-associated fibroblasts CAF and the patient-derived primary tumor cells PDC comprises the following steps: taking a lymphoma patient lymphoid tissue sample (such as a lymph node biopsy sample), washing and cutting, adding digestive enzymes for treatment, then centrifuging, discarding the supernatant, and resuspending the cells with human tissue washing solution; then, by taking advantage of the characteristics of fibroblasts adhering to the wall and lymphoma cells growing in suspension, the patient-derived tumor-associated fibroblasts CAF and the patient-derived primary tumor cells PDC are enriched, expanded and separated.

[0012] Preferably, the formula of the digestive enzymes is: 1 mL of 1 mg / mL DNase + 5 mL of 2 mg / mL collagenase IV + 10 μL of 10 mM Y-27632 (ROCK inhibitor), diluted to 10 mL with human tissue washing solution.

[0013] Preferably, the formula of the human tissue washing solution is: RPMI 1640 medium + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 100 μg / mL Primocin (primary cell antibiotic) + 1% BSA (bovine serum albumin).

[0014] The patient-derived tumor-associated fibroblasts CAF are frozen in a patient-derived tumor-associated fibroblast CAF freezing solution for subsequent use; the formula of the patient-derived tumor-associated fibroblast CAF freezing solution is: DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12) + 1% PS (penicillin-streptomycin) + 20% FBS (fetal bovine serum) + 10% DMSO (dimethyl sulfoxide); The patient-derived primary tumor cells PDC are frozen in a primary freezing solution for subsequent use; the formula of the primary freezing solution is: DMEM (Dulbecco's Modified Eagle Medium) + 1% PS (penicillin-streptomycin) + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 50% FBS (fetal bovine serum) + 10% DMSO (dimethyl sulfoxide) + 10 μM Y-27632 (ROCK inhibitor); Further, the lymphoma cells are mixed with the engineered stromal cells ESC or the patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1 (preferably at a ratio of 10:1-5:1, and more preferably at a ratio of 10:1), and inoculated in a lymphoma organoid culture medium (OCM) for in vitro co-culture.

[0015] Further, the in vitro co-culture is performed by any of the following ways: (a) 2.5D co-culture: lymphoma cells are seeded on engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1, and cultured in 2.5D in OCM medium; (b) 3D ultra-low attachment culture: lymphoma cells are mixed with ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1, and seeded in 96-well flat-bottom ultra-low attachment multi-well plates for suspension culture in OCM medium; (c) 3D Matrigel co-culture: lymphoma cells are mixed with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1, and the resulting cell suspension is embedded with Matrigel at a volume ratio of 1:1 to form a three-dimensional structure for culture; (d) 3D agarose microarray co-culture: lymphoma cells are mixed with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1 in OCM medium, and seeded into a pre-formed agarose microwell array for culture after standing for 10-30 min; (e) Other commercial microwell plates that can be used for 50 μm~500 μm microsphere culture, including but not limited to 96Well plate (SUN Bioscience), Millicell® Microwell Plates, MicroTissues® 3D PetriDish®, AggreWell™ Microwell Plates (STEMCELL), etc.

[0016] Preferably, the formula of the OCM medium is: DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12) + 10% FBS (fetal bovine serum) + 1% PS (penicillin-streptomycin) + 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 10 ng / mL IL-4 (interleukin-4).

[0017] Preferably, the in vitro co-culture is performed by 2.5D or 3D method.

[0018] Preferably, the culture conditions are: 37℃, 5% CO2.

[0019] In a second aspect, the present application provides the use of the lymphoma organoids obtained according to the method as a screening model for anti-tumor drugs.

[0020] In a third aspect, the application provides use of the lymphoma organoids obtained according to the method in anti-tumor drug screening and individualized drug sensitivity detection.

[0021] By means of the technical scheme, the application has at least the following advantages and beneficial effects: The application provides an in vitro culture method of patient-derived B cell lymphoma organoids, which has the advantages of adding stromal cells to the in vitro 2.5D and 3D culture system, providing cell cross-talk between tumor cells and stromal cells, better simulating the in vivo tumor microenvironment of lymphoma in vitro, and significantly prolonging the in vitro culture survival time of patient-derived lymphoma cells. Meanwhile, the application not only provides a co-culture method of commercial cell lines as stromal cells, but also provides extraction, culture and co-culture methods of patient-derived primary tumor-associated fibroblasts CAF, which can further simulate the tumor microenvironment and support the in vitro survival of lymphoma cells. The in vitro co-culture system provided by the application introduces engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF, which not only stably improves the in vitro survival of lymphoma cells, but also provides more realistic drug sensitivity. Compared with a single culture system, the system can to some extent avoid the problems of single cell type, low cell viability and short survival time, which lead to experimental drug sensitivity results higher than the actual drug sensitivity. The 3D co-culture and microarray culture provided by the application provide a real and reliable in vitro model for drug sensitivity testing of patient-derived cells, and provide a new tool for lymphoma drug screening. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The left graph shows the ratio of DLBCL PDXC and ESC at day 10 of culture in the preferred embodiment of the application. The right graph shows the PI staining to identify the death rate of DLBCL PDXC in the preferred embodiment of the application.

[0023] Figure 2 The CellTrace™ far-red cell proliferation reagent is used to label the proliferation of tumor cells in the DLBCL PDX organoid (DLBCL PDXO) in the preferred embodiment of the application.

[0024] Figure 3 The patient-derived CAF transfected with lentivirus SV40T antigen is observed by fluorescence microscope in the preferred embodiment of the application. Green fluorescent protein (GFP) positive indicates successful transfection. Scale bar, 200 μm.

[0025] Figure 4Figure 6 shows the images of DLBCL PDXC and patient-derived CAF 2.5D co-culture taken by high content imaging analyzer in the preferred embodiment of the present application. Left panel: bright field, right panel: fluorescence merged image. Blue, nucleus; red, Ki67; green, CAF. Scale bar, 400 μm.

[0026] Figure 5 Figure 7 shows the statistical graphs of different cell numbers and proportions in the DLBCL PDXC and CAF 2.5D co-culture system in the preferred embodiment of the present application. Left panel: CAF, Ki67-positive DLBCL and Ki67-negative DLBCL cell number statistics, right panel: Ki67-positive DLBCL and Ki67-negative DLBCL proportion.

[0027] Figure 6 Figure 8 shows the cell distribution of DLBCL PDXO observed by confocal microscope in the preferred embodiment of the present application. Blue, DAPI-labeled nucleus; red, CD20-labeled PDXC; green, ESC expressing green fluorescent protein (GFP). Scale bar, 200 μm.

[0028] Figure 7 Figure 9 shows the changes in cell viability during the cultivation of 3D ultra-low attachment co-cultured PDO verified by flow cytometry in the preferred embodiment of the present application.

[0029] Figure 8 Figure 10 shows the changes in cell proportion during the cultivation of 3D ultra-low attachment co-cultured PDO verified by flow cytometry in the preferred embodiment of the present application.

[0030] Figure 9 Figure 11 shows the histological identification of 3D ultra-low attachment co-cultured PDXO and its corresponding PDX tissue in the preferred embodiment of the present application. Identification indicators include CD79, CD20, CD3, and Ki67. Scale bar, 500 μm.

[0031] Figure 10 Figure 12 shows the PDO taken by high content imaging analyzer in the preferred embodiment of the present application. Blue, labeled nucleus; red, dead cells; green, ESC expressing green fluorescent protein (GFP).

[0032] Figure 11 Figure 13 shows the PDO of the experimental group and the control group taken by high content imaging analyzer in the preferred embodiment of the present application.

[0033] Figure 12 Figure 14 shows the analysis of the reaction of PDO to different drug low, medium, and high doses in the preferred embodiment of the present application.

[0034] Figure 13High-content imaging photos of PDXC and MRC5-LB cell 3D agarose microarray co-cultured PDO for use in the drug sensitivity test of Xidabemide in the preferred embodiment of the present application, the top is the bright field photos of the control group and the experimental group, and the bottom is the fluorescence combined photos of the control group and the experimental group, each group is 6 repeats. Blue, cell nucleus; red, dead cells; green, MRC5-LB. Scale, 400 μm.

[0035] Figure 14 Statistical analysis chart of the average fluorescence intensity values of different channels of the control group and the experimental group for PDXC and MRC5-LB cell 3D agarose microarray co-cultured PDO for use in the drug sensitivity test of Xidabemide in the preferred embodiment of the present application. The statistical difference between the two groups is evaluated by data analysis through unpaired T test, P<0.05, P<0.01, there is a significant statistical difference.

[0036] Figure 15 Image of mantle cell lymphoma patient-derived primary cells (MCL PDC) and MRC5-LB co-cultured to construct mantle cell lymphoma patient-derived organoids (MCL PDO) after administration of venetoclax, taken by the high-content imaging analyzer in the embodiment of the present application. Blue, cell nucleus; red, dead cells; green, MRC5-LB. Scale, 400 μm.

[0037] Figure 16 Statistical analysis of the total fluorescence intensity ratio of the MCL PDO images taken by the high-content imaging analyzer in the embodiment of the present application, the statistical difference between multiple groups is evaluated by data analysis through one-way ANOVA, P<0.05, P<0.01, there is a significant statistical difference.

[0038] Figure 17 Hematoxylin-eosin (H&E) staining image of a representative MCL PDO in the embodiment of the present application. Scale, 50 μm. DETAILED DESCRIPTION

[0039] The present application aims to provide a method for constructing B cell lymphoma patient-derived organoids (BCL PDO), and the main technical problem to be solved is to enable B cell lymphoma cells to stably survive in a three-dimensional structure with multiple cell components in vitro through co-culture with engineered stromal cells ESC or patient-derived tumor-related fibroblasts CAF; this model not only better simulates the growth state of B cell lymphoma in vivo, but also provides a research platform for individualized drug sensitivity and large-scale drug screening of B cell lymphoma.

[0040] The application adopts the following technical solutions: The application mainly relates to: BCL PDO construction including ESC establishment and function evaluation, PDX tissue processing, patient-derived lymphoma tissue processing and patient-derived CAF separation, BCL PDO and PDX organoid (PDXO) construction and identification, BCL PDO-based drug sensitivity detection and the like steps.

[0041] 1. Establishment and function evaluation of engineered stromal cells ESC Construct a lentivirus vector (pSLenti-SFHEGFP-P2A-Puro-CMV-TNFSF13B-P2A-CD40LG-WPRE), stably transfect GFP, CD40L and BAFF into fibroblasts or stromal cells (such as human embryonic lung fibroblasts MRC5) to obtain ESC (named as MRC5-LB); Cultivate the ESC in a 6-well plate, inoculate the BCL PDXC after the cells adhere to the wall, detect the BCL PDXC activity and its proliferation at multiple time points during the co-culture process by using flow cytometry, and identify the growth support effect of the ESC cells on the BCL PDXC.

[0042] 2. PDX tissue processing and PDXC acquisition Obtain BCL PDX mouse tumor tissue, store in a tissue preservation solution, and transport on ice. The formula of the tissue preservation solution is: RPMI 1640 culture medium or DMEM culture medium (Dulbecco's modified Eagle's medium) + 1%-2% PS (penicillin-streptomycin) + 100 μg / mL Primocin (primary cell antibiotic). Wash the tissue with human tissue washing solution for 3-5 times, trim the tissue, and discard fat, fascia and necrotic tissue. The formula of the human tissue washing solution involved in the above method is: RPMI 1640 culture medium + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 100 μg / mL Primocin (primary cell antibiotic) + 1% BSA (bovine serum albumin). Mechanically dissociate the tissue on ice as quickly as possible, cut it into pieces as much as possible, add human tissue washing solution, collect the supernatant through a 70 μm sieve, collect into a centrifuge tube, and place on ice to wait for centrifugation. The centrifugation condition is 300-400 g centrifugation for 5 min at 4°C or room temperature. Digestion with digestive enzymes at 37°C, 10-20 min each time, observe the cell shedding, depending on the tissue digestion, can be digested 1-3 times, each time using 2-3 mL of digestive enzymes, each time collect the supernatant after fully mixed and natural sedimentation through 70 μm screen, collect into centrifuge tube, placed on ice to wait for centrifugation, centrifugation conditions are 300-400 g for 5 min, 4°C or room temperature; The digestive enzyme formula involved in the above method is: 1 mL 1 mg / mL DNAase + 5 mL 2 mg / mL collagenase IV + 10 μM Y-27632 (ROCK inhibitor), diluted to 10 mL with human tissue washing solution; The obtained cell suspension is centrifuged at 300-400 g for 5 min at 4°C or room temperature, and the supernatant is discarded; Observe the color of the cell sediment obtained by centrifugation, if the red blood cell content is high, red blood cell lysis treatment can be performed, if not, resuspend and count the cells after resuspension; The method of red blood cell lysis involved in the above method is: According to 1-2 times the volume of the cell sediment, add room temperature red blood cell lysis solution, resuspend the sediment after shaking the tube, mix well, and then stand at room temperature for 1-2 min, add 6-10 times the volume of lysis solution to human tissue washing solution; centrifuge at 300-400 g for 5 min at 4°C or room temperature, carefully aspirate the supernatant without disturbing the sediment; resuspend the cells with the same volume of human tissue washing solution, centrifuge at 300-400 g for 5 min at 4°C or room temperature, and carefully aspirate the supernatant without disturbing the sediment.

[0043] 3. Processing of patient-derived lymphoma tissue and isolation of patient-derived CAF (1) Processing of patient-derived lymphoma tissue After obtaining the lymph node biopsy sample of a B cell lymphoma patient, take a photo to record the sample information; Transfer the sample to a sterile culture dish, wash the sample with human tissue washing solution, wash 3-5 times; trim, discard necrotic tissue, fat and fascia; rinse the tissue with human tissue washing solution again, transfer the tissue to a new sterile culture dish, take a photo to record the size of the tissue and record the weight of the tissue using a balance; The human tissue washing solution in the above method is the same as described above; Cut the tissue in a sterile culture dish, take an appropriate amount of tissue and freeze it in a commercial cryopreservation solution (STEMCELL CS10), about 1-3 mm in size 3 ; Transfer the tissue into a 1.5 mL EP tube, continue to cut into small pieces on ice, add human tissue wash solution, aspirate the supernatant through a 70 μm mesh, repeat several times to collect the cells by adding human tissue wash solution until the supernatant is no longer turbid, collect the mechanically cut cells into a 15 mL centrifuge tube, and place on ice; Transfer the cut tissue into a 5 mL centrifuge tube, try to aspirate the supernatant, add 2-4 mL of digestive enzyme, shake at 37°C for 10-20 min, after digestion, mix the tissue suspension well, let it settle naturally, aspirate the supernatant through a mesh, collect the cells into a 15 mL centrifuge tube, and place on ice, a total of 3 times of digestion; The digestive enzyme involved in the above method is the same as the foregoing; Observe the state of tissue digestion, if the digestion is not thorough, add 2-3 mL of TrypLE™ Express (trypsin substitute) at 37°C for 10-20 min, collect the cells obtained by digestion into a 15 mL centrifuge tube, and place on ice; Transfer the digested tissue to a mesh, grind the tissue in the mesh with the plunger of a 1 mL syringe, rinse the mesh with human tissue wash solution, collect as many cells as possible that fall off, collect the ground cells into a 15 mL centrifuge tube, and place on ice; Centrifuge the cell suspension collected by mechanical dissociation and enzymatic digestion at 300-400 g for 5 min, discard the supernatant; Perform red blood cell lysis as appropriate according to the proportion of red blood cells, and the operation is the same as the foregoing; Resuspend the cells with DMEM medium (containing 1% penicillin-streptomycin), stain with trypan blue, and count the cells using a cell counter to detect cell density and viability; Adjust the cell density, freeze the PDCs using a primary freezing solution, and the formula of the patient-derived primary freezing solution involved is: DMEM (Dulbecco's Modified Eagle Medium) + 1% PS (penicillin-streptomycin) + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 50% FBS (fetal bovine serum) + 10% DMSO (dimethyl sulfoxide) + 10 μM Y-27632 (ROCK inhibitor); (2) Isolation of patient-derived CAFs Take a portion of PDC cells (about 5E6) in a 6 cm sterile culture dish, take advantage of the characteristics of fibroblast cells adhering and lymphoma cells growing in suspension, and enrich and expand patient-derived CAFs.

[0044] The formula of the patient-derived CAF culture medium involved in the above method is: DMEM / F12 (Dulbecco Modified Eagle Medium / Ham's F-12) + 10% FBS (Fetal Bovine Serum) + 1% PS (Penicillin-Streptomycin) + 10 ng / mL bFGF (Basic Fibroblast Growth Factor) + 10 mM HEPES (N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 2 mM GlutaMAX™ Supplement (L-Glutamine Substitute) + 1 mM Sodium Pyruvate + 0.1 mM Non-essential Amino Acids; (3) Constructing an immortalized CAF Using a lentiviral vector, SV40T antigen (NCBI gene number: NC_001669-SV40gp6) and GFP were transfected into CAF; Patient-derived CAF culture medium was supplemented with appropriate selection and maintenance concentrations of puromycin to screen and maintain transfected patient-derived CAF. Flow cytometry was used to detect co-transfected GFP-expressing positive cells to determine the proportion of successfully transfected cells. Screening was continued until the proportion of successfully transfected cells reached more than 98%. Cells were cryopreserved using patient-derived CAF cryopreservation solution for subsequent co-culture and mechanistic studies.

[0045] The formulation of the patient-derived CAF cryopreservation solution involved in the above method is as follows: DMEM / F12 (Dulbecco Modified Eagle Medium / Ham's F-12) + 1% PS (Penicillin-Streptomycin) + 20% FBS (Fetal Bovine Serum) + 10% DMSO (Dimethyl Sulfoxide). 4. Construction and Qualification of BCL PDO or PDXO (1) Construction of BCL PDO or PDXO Collect lymphoma cells (PDXC or PDC) and co-culture them with stromal cells (ESC or patient-derived CAF) for 2.5D or 3D. stromal cell preparation: Resuscitate stromal cells 3 days in advance, culture until 90% confluence, digest and centrifuge the cells, resuspend in OCM and count the cells. (a) 2.5D co-culture: Lymphoma cells were seeded onto stromal cells at a ratio of stromal cells to lymphoma cells of 1:10 and cultured using OCM at 37°C and 5% CO2. (b) 3D ultra-low adsorption co-culture: The two types of cells were mixed in OCM at a ratio of stromal cells: lymphoma cells = 1:10 to obtain a cell suspension, which was then seeded in a 96-well flat-bottom ultra-low adsorption multi-well plate and cultured in suspension at 37°C and 5% CO2. (c) 3D Matrigel co-culture: mix the two cells in OCM cell suspension at a ratio of stromal cells:lymphoma cells = 1:10, mix the cell suspension with Matrigel at a volume ratio of 1:1, point 10 μL / well in a 96-well plate, incubate at 37°C, 5% CO2 for 15-30 min, after the Matrigel solidifies, add 200 μL OCM per well for culture; (d) 3D agarose microarray co-culture: mix the two cells in OCM cell suspension at a ratio of stromal cells:lymphoma cells = 1:10, inoculate into a pre-formed agarose microarray, after standing for 10 to 30 min, culture at 37°C, 5% CO2; Observe the growth of 2.5D and 3D co-cultured cells (intercellular interaction and relative position of lymphoma cells and stromal cells, whether the lymphoma cells grow into spheres) during culture.

[0046] (2) Identification of multiple cell components in the co-culture system Observe the cell growth state every day, and use flow cytometry to detect cell proportion changes and cell viability on days 0, 1, 3, 6, 9, 12, 15, 18, and 21 of culture; The cell surface markers selected by flow cytometry are: T cells (CD3, CD4, CD8), B cells (CD 19, CD20), ESC (GFP), and zombie dye to represent cell death and life; Detect the proportion and viability changes of different cells in the co-culture system at different time points; (3) Histological identification of BCL PDO or PDXO Fix the BCL PDO or PDXO obtained by co-culture in 4% paraformaldehyde, perform H&E staining and immunohistochemical staining, and label the biomarkers including CD20, CD79, CD3, and Ki67; Compare the H&E and IHC results of patient tumor tissue and PDO or PDX tissue and PDXO to verify histological consistency; 5. Drug sensitivity detection based on BCL PDXO and BCL PDO Closely observe the growth of BCL PDO or BCL PDXO in the 3D co-culture group in the 96-well plate, and after observing the cell aggregation into spheres, proceed to the subsequent drug screening experiment; Set up low, medium, and high dose groups and a blank control group, use OCM to prepare the experimental group drugs at the corresponding concentrations, discard the original culture medium, and add 200 μL of culture medium containing drugs per well; After 48-72 h of drug treatment, the drug responsiveness of BCL PDO / PDXO was assessed using a high-content imaging analyzer. Dead cells were labeled with PI dye and cell nuclei were labeled with Hoechst-33342 before imaging analysis. The PI and Hoechst-33342 staining methods mentioned above are explained below: The concentration range of PI is 0.1-10 µM. The ideal staining effect is to stain only the cell nucleus without staining the cytoplasm, which is the optimal working concentration; the concentration of Hoechst-33342 is 20 μM. The average area, PDXO / PDO count, channel area, and total fluorescence intensity of PDXO / PDO were analyzed using the built-in image analysis software of a high-content imaging analyzer to evaluate the drug reactivity of PDXO / PDO.

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0048] Example 1: ESC Establishment and Functional Evaluation A lentiviral vector was constructed, and GFP, CD40L and BAFF were stably transfected into human embryonic lung fibroblasts MRC5 to obtain MRC5-LB. The formula for MRC5-LB medium is: DMEM (Dulbecco modified Eagle medium) + 1% PS (penicillin-streptomycin) + 10% FBS (fetal bovine serum). 2.5D Co-culture: MRC5-LB cells were cultured in 6-well plates. After cell adhesion, DLBCL PDXC cells were seeded. The viability and proliferation of PDXC cells were detected and statistically analyzed by flow cytometry at 1, 3, 5, 7 and 10 days after culture to identify the supporting role of MRC5-LB cells in the growth of DLBCL PDCs.

[0049] Depend on Figure 1 It was found that on day 10 of culture, the ratio of DLBCL PDXC to MRC5-LB cells was approximately 6:4, and PI staining indicated that the viability of DLBCL PDXC was ~99%; Figure 2 As shown, the proliferation of DLBCLPDXC was marked using CellTrace™ far-infrared cell proliferation reagent. TM As the dilution percentage gradually increased with culture, it was observed that DLBCL PDXC maintained proliferation.

[0050] Example 2: Processing of PDX tissue and acquisition of PDXC Euthanize the DLBCL PDX mouse, alcohol disinfect the skin at the tumor site, dissect the tumor completely, and store in RPMI 1640 medium + 1% PS (penicillin-streptomycin) + 100 pg / mL Primocin (primary cell antibiotic) on ice; Wash the tissue with human tissue wash solution for 3 times, trim the tissue, and discard the fat, fascia, and necrotic tissue. The formula of the human tissue wash solution is: RPMI 1640 medium + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 100 pg / mL Primocin (primary cell antibiotic) + 1% BSA (bovine serum albumin). After cutting the tissue into small pieces (about 1 mm 3 ) in a sterile petri dish, transfer to a centrifuge tube, shred the tissue on ice as much as possible, add human tissue wash solution and mix well, let it settle naturally, collect the supernatant through a 70 pm sieve, collect into a 15 mL centrifuge tube, and place on ice to wait for centrifugation. Add digestive enzymes for digestion at 37°C. The formula of the digestive enzymes is: 1 mL of 1 mg / mL DNase + 5 mL of 2 mg / mL collagenase IV + 10 mM Y-27632 (ROCK inhibitor), diluted to 10 mL with human tissue wash solution; use 3 mL of digestive enzymes each time, digest for 15 min, a total of 3 times of digestion, collect the supernatant after each time of mixing well and natural settling through a 70 pm sieve, collect into a centrifuge tube, and place on ice to wait for centrifugation. Centrifuge the obtained cell suspension at 400 g for 5 min at room temperature, discard the supernatant, resuspend with OCM for cell counting and viability detection, and obtain PDXCs.

[0051] The formula of the OCM is: DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12) + 10% FBS (fetal bovine serum) + 1% PS (penicillin-streptomycin) + 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 10 ng / mL IL-4 (interleukin-4).

[0052] Example 3. Processing of patient-derived lymphoma tissue and isolation of patient-derived CAFs After obtaining the patient lymph node biopsy sample by surgery, store it in tissue preservation solution, transport it to the laboratory on ice, and record the patient sample information. Transfer the sample to a sterile petri dish and wash the sample with human tissue wash solution for at least 3 times. The formula of human tissue wash solution is the same as described in Example 2. Trim the tissue with autoclaved ophthalmic scissors and ophthalmic forceps, discard the necrotic tissue, fat and fascia; Rinse the tissue with human tissue wash solution, transfer the tissue to a new sterile petri dish, take a photo to record the size of the tissue and record the weight of the tissue with a balance; Cut the tissue into small pieces in a sterile petri dish, take an appropriate amount of tissue and freeze it in a commercial freezing solution (STEMCELL CS10), about 1 mm in size 3 ; Transfer the tissue to a 1.5 mL EP tube with a Pasteur pipette (fully rinsed with human tissue wash solution), continue to cut it on ice, add human tissue wash solution, mix well, let it settle naturally, aspirate the supernatant through a 70 μm sieve, and repeat the addition of human tissue wash solution until the supernatant is no longer turbid. Collect the mechanically cut cells into a 15 mL centrifuge tube, name it Fraction 1, and place it on ice; Transfer the cut tissue to a 5 mL centrifuge tube with a Pasteur pipette, try to aspirate the supernatant, add 3 mL of digestive enzyme, and shake at 37°C for 15 min. The formula of the digestive enzyme is described in Example 2. After digestion, mix the tissue suspension well, let it settle naturally, aspirate the supernatant through a sieve, and collect the cells obtained from the first digestion into a 15 mL centrifuge tube, name it Fraction 2, and place it on ice. Repeat the digestion for a total of 3 times, and collect Fractions 3 and 4 in sequence, and place them on ice. Observe the state of tissue digestion. If the digestion is not thorough enough, add 2 mL of TrypLE™ Express (trypsin substitute) to 37°C for 20 min, collect the cells obtained from the fourth digestion into a 15 mL centrifuge tube, name it Fraction 5, and place it on ice. Transfer the digested tissue to a sieve, grind the tissue with a 1 mL syringe piston, rinse the sieve with human tissue wash solution, and collect as many cells as possible that fall off. Collect the ground cells into a 15 mL centrifuge tube, name it Fraction 6, and place it on ice. Centrifuge the collected cell suspension at 400 g for 5 min, discard the supernatant; Perform red blood cell lysis operation according to the amount of red blood cells. Resuspend the cells with an appropriate amount of red blood cell lysis solution, lyse at room temperature for 1-2 min, centrifuge at 400 g for 5 min, and discard the supernatant; Resuspend the cells with human tissue wash solution, centrifuge at 400 g for 5 min, and discard the supernatant; Resuspend the cells with OCM, mix well, take 10 μL cell suspension, mix well with 2x trypan blue solution, and use a cell counter to detect cell density and viability. Adjust the cell density, and freeze the PDCs using the primary freezing solution. The formula of the primary freezing solution is the same as described above. Take about 5E6 PDCs in a sterile culture dish, and use the characteristics of fibroblast adhesion and lymphoma cell suspension growth to enrich and expand patient-derived CAFs. The culture medium for patient-derived CAFs is as follows: DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12) + 10% FBS (fetal bovine serum) + 1% PS (penicillin-streptomycin) + 10 ng / mL bFGF (basic fibroblast growth factor) + 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) + 2 mM GlutaMAX™ supplement (L-glutamine substitute) + 1 mM sodium pyruvate + 0.1 mM non-essential amino acids. Immortalization of CAFs: Transfect SV40T antigen and GFP into CAFs using lentivirus, add an appropriate concentration of puromycin to the culture medium for screening and maintenance of culture, and observe the expression of green fluorescent protein (GFP) in cells under a fluorescence microscope. Figure 3 Freeze the cells for subsequent co-culture and mechanism research.

[0053] Example 4: Identification of the proliferation of lymphoma cells co-cultured with CAFs 1. Construction of a 2.5D co-culture system based on CAFs (1) Preparation of patient-derived CAFs: Recover the immortalized CAFs one day in advance, inoculate 1E4 cells / well in a ultra-thin glass bottom 96-well plate (Cellvis P96-1.5H-N), and culture using the patient-derived CAF culture medium (as described in Example 3) so that the cell confluence rate is 80%-90% the next day. (2) Tumor cell inoculation: Inoculate PDXC at a ratio of CAF:PDXC=1:5, 5E4 cells / well, and use OCM (as described in Example 2) for culture. Observe the growth of DLBCL PDXC during the culture process, and observe the cell-cell interaction and relative position of tumor cells and stromal cells. 2. Detection of Ki67 expression as a proliferation indicator of DLBCL PDXC On the 3rd day of co-culture, perform Ki67 immunofluorescence staining, and the specific method is as follows: (1) Fixation, membrane rupture: carefully aspirate the culture medium, add 50 μL of 4% paraformaldehyde fixing solution to each well, fix the cells at room temperature for 10 min, aspirate the fixing solution, add 50 μL of 0.1% Triton X-100 to each well, and rupture the membrane at room temperature for 5 min, aspirate the membrane rupture solution, and wash with PBS for 3 times, 5 min each time; (2) Blocking: add 100 μL of 1% BSA to each well, and block at room temperature for 1 h; (3) Aspirate the blocking solution, add 50 μL of Ki67 rabbit monoclonal antibody (1:250 dilution) to each well, and incubate at room temperature for 1 h; (4) Add 100 μL of PBST washing buffer to each well, and wash at room temperature for 3 times, 5 min each time. The formula of the PBST washing buffer is: PBS buffer + 0.1% Tween-20; (5) Add 50 μL of 2 μg / mL goat anti-rabbit IgG (Alexa Fluor® 647) to each well, and incubate at room temperature for 1 h; (6) Add 100 μL of PBST washing buffer to each well, and wash at room temperature for 3 times, 5 min each time; (7) Add 50 μL of cell nucleus staining solution (Hoechst-33342) to each well, and stain at room temperature for 5 min, aspirate the staining solution, and add 50 μL of PBS to each well; (8) Use a high-content analyzer (Perkin Elmer Operetta) to perform imaging analysis, and take red (Ki67), green (CAF) and blue (nucleus) fluorescence images, respectively. The imaging results are shown in FIG. 6A-6C. The number of CAF and DLBCL PDXC nuclei was counted, respectively, and the number of Ki67-positive DLBCL PDXC was counted (FIG. 6D). The results show that the average proportion of Ki67-positive DLBCL PDXC co-cultured with CAF is 45.63%, and it can be seen that the co-culture system based on CAF can promote the proliferation of DLBCL PDXC. Figure 4 Figure 5

[0054] Example 5 Construction of PDXO or PDO 1. Preparation of stromal cells The ESC used in this example is MRC5-LB cells; The MRC5-LB cells were recovered 3 days in advance, and were cultured to a confluence rate of 90%. The stromal cells were digested with trypsin (0.25%, containing 2.21 mM EDTA), and the cells were collected by centrifugation. After resuspension of OCM, cell counting was performed; 2. Mixing of stromal cells and lymphoma cells (1) 2.5D co-culture: ​​1 6 cm sterile petri dish of MRC5-LB cells (fusion rate 90%), total cell number about 3E5; Inoculate at a ratio of MRC5-LB cells: PDC = 1:10, a total of 3E6 lymphoma cells, OCM volume about 3 mL; (2) 3D ultra-low adsorption co-culture: Mix MRC5-LB cells and lymphoma cells in OCM at a ratio of MRC5-LB cells: lymphoma cells = 1:10 to obtain a cell suspension, and inoculate 200 μL / well in a 96-well ultra-low adsorption flat-bottom multi-well plate to perform suspension culture; (3) 3D Matrigel co-culture: Mix MRC5-LB cells and lymphoma cells at a ratio of MRC5-LB cells: lymphoma cells = 1:10 to obtain a cell suspension, and place on ice to keep the cell suspension at 4°C; 3E4 cells (PDC + MRC5-LB) per well, a total of 60 wells, prepare 64 well cell suspension and Matrigel mixture, 320 μL Matrigel is added to 320 μL cell suspension (total cell number is 1.92E6), and point 10 μL / well in an ultra-thin glass bottom 96-well plate (Cellvis P96-1.5H-N), and incubate at 37°C, 5% CO2 for 15 min, and after the Matrigel solidifies, add 200 μL OCM per well for culture, and add PBS to the edge wells to prevent the culture medium from evaporating too quickly; (4) 3D agarose microarray co-culture: Mix PDC and ESC or patient-derived CAF at a ratio of 10:1 in OCM, and inoculate into a preformed agarose microarray, and culture after standing for 10 to 30 min; The formula of OCM is the same as described in Example 2; Observe the growth of 2.5D and 3D co-cultures during the culture process, and use a confocal microscope to observe the DLBCL PDXO of 3D ultra-low adsorption co-culture, and the results are shown in Figure 6 .

[0055] Example 6 Viability and cell proportion identification of 3D ultra-low adsorption co-cultured PDO Observe the cell growth state every day, and use flow cytometry to detect the cell viability and cell proportion change on day 0, 1, 3, 6, 9, 12, 15, 18, and 21 of the culture; The cell surface markers selected by flow cytometry are: CD3, CD4, CD8, CD 19, CD20, and zombie dye is used to characterize cell death and life; The flow cytometry sample processing process is as follows: using trypsin (0.25%, containing 2.21 mM EDTA) to digest the cells at 37°C for 1-2 min, after stopping the digestion, blowing evenly and collecting the cells in a 1.5 mL EP tube, centrifuging at 400 g for 5 min, washing with PBS once, resuspending the cells with 50 μL PBS after centrifugation, adding Human Fc Block (1:20) and zombie (1:200), incubating at room temperature for 15 min in the dark, mixing 3-5 times during the incubation, at the end of the incubation, adding 1 mL PBS, centrifuging at 400 g for 5 min, discarding the supernatant; Resuspending the cells with 50 μL PBS (containing 1% FBS), adding CD3, CD4, CD8, CD19, CD20 antibodies (1:200), incubating at room temperature for 15 min in the dark, mixing 3-5 times during the incubation, at the end of the incubation, adding 1 mL PBS, centrifuging at 400 g for 5 min, discarding the supernatant; Resuspending the cells with 200 μL PBS, filtering the cell suspension with a 150 μm sieve into a flow tube, and detecting on the machine, collecting 10,000 single cells as the collection endpoint.

[0056] The results of using flow cytometry to detect cell viability and cell proportion change are shown in Figure 7 and Figure 8 It can be seen that the PDO viability is maintained at more than 70% within 21 days, and the cell proportion of PDO is stable within 10 days, which can be used for subsequent experiments.

[0057] Example 7 Histological identification of PDO 1. Sectioning (1) Fixation: Collecting 3D ultra-low adsorption co-cultured PDXO, fixing with 4% paraformaldehyde, and fixing for 1-2 h; (2) Dehydration: Dehydrating with gradient alcohol, 75% for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h, 100% (I) for 30 min, and 100% (II) for 30 min.

[0058] (3) Transparency: Xylene (I) for 10 min, xylene (II) for 10 min.

[0059] (4) Wax immersion: Wax immersion (I) for 1 h, and wax immersion (II) for 1 h.

[0060] (5) Embedding: Placing the wax-immersed tissue in an embedding frame for embedding. First, add a small amount of melted wax liquid to the embedding frame, and before it solidifies, place the tissue, then add enough wax liquid to embed the tissue block, and place it on a-20°C freezing table to fully solidify the wax block.

[0061] (6) Slicing: The wax block was solidified enough, fixed on the automatic slicer, and cut into 4-5 μm thin slices.

[0062] 2. Hematoxylin-eosin (H&E) staining (1) De-waxing: The glass slide was baked on the slide baker for 1 h, xylene (I) for 10 min; xylene (II) for 10 min; 100% ethanol (I) for 5 min; 100% ethanol (II) for 5 min.

[0063] (2) Hydration: 95%, 85%, 75% ethanol were sequentially hydrated for 5 min; distilled water was used for hydration.

[0064] (3) Staining: hematoxylin staining for 5 min, water flushing; hydrochloric acid alcohol differentiation for 1 min, water flushing; eosin staining for 1 min.

[0065] (4) Dehydration: 85%, 95%, 100%, 100% gradient alcohol was sequentially dehydrated for 1 min.

[0066] (5) Transparency.

[0067] (6) Mounting: neutral gum was added dropwise for mounting.

[0068] 3. Immunohistochemical staining (1) De-waxing and hydration.

[0069] (2) Antigen repair: the glass slide was placed in a glass container containing citrate solution (1x), the solution was completely immersed in the slice, and was heated in a microwave oven, after boiling at high fire, the solution was boiled at medium-low fire for 10 min, the solution was kept boiling within 10 min, and the slide was washed with PBS for 3 times, 5 min each time.

[0070] (3) Blocking endogenous peroxidase: the slide was completely immersed in 3% H202 solution, and was blocked at room temperature for 30 min, and then was washed with PBS for 3 times, 5 min each time.

[0071] (4) Blocking endogenous antigen: 5% BSA antigen blocking solution was prepared by using 0.1% PBST, and was blocked at room temperature for 1 h.

[0072] (5) Primary antibody incubation: an appropriate amount of pre-prepared primary antibody working solution was added, and was incubated in a wet box at 4°C overnight, the primary antibody labeled biomarker included CD20, CD79, CD3, and Ki67.

[0073] (6) Secondary antibody incubation: the primary antibody working solution was shaken off, and was washed with PBS for 3 times, the corresponding species of HRP labeled secondary antibody working solution was added, and was incubated in a wet box at room temperature for 1 h.

[0074] (7) DAB color developing solution color developing: spin dry the secondary antibody working solution, and drop a proper amount of DAB color developing solution.

[0075] (8) Color developing under a microscope and timely terminating the staining with running water.

[0076] (9) Hematoxylin re-staining: immerse the rinsed paraffin section into hematoxylin staining solution for 10-20 seconds, and then wash the hematoxylin staining solution with running water.

[0077] (10) Dehydrating, transparentizing, and mounting.

[0078] The section was observed using an optical microscope, and the results are shown in Figure 9 It can be seen that the 3D ultra-low adsorption co-cultured PDXO and the corresponding PDX tissue are consistent at the histological level.

[0079] Application of PDO in drug screening The growth of the 3D co-cultured PDO was observed under a microscope every day, and after the cells aggregated into spheres (24-48 h), the subsequent drug screening experiment was performed; Screened drugs and doses: according to the half inhibitory concentration (IC50) of the cell experiment concentration, three doses of low, medium and high were set, i.e., rituximab (1 μg / mL, 10 μg / mL, 20 μg / mL), geftifimab (10 ng / mL, 100 ng / mL, 1 μg / mL), chidamide (1 μM, 5 μM, 10 μM), SHR2554 (2.5 μM, 12.5 μM, 25 μM), and ibritumomab (100 nM, 1 μM, 10 μM); Drug preparation: the experimental group drugs of corresponding concentrations were prepared using OCM (as described in Example 2); Drug treatment: the original culture medium in the 96-well plate was discarded, and the culture medium containing the drug was added; The growth state of the organoids was observed under a microscope every day after the drug treatment, and the PDO drug response was evaluated using a high-content imaging analyzer after 72 h; Staining treatment: 50 μL / well of 1.5 μM PI solution was added, and the staining was performed at room temperature in the dark for 15 min, and the PI staining solution was aspirated; 50 μL / well of 20 μM Hoechst-33342 was added, and the incubation was performed at room temperature in the dark for 10 min, and the staining solution was not discarded, and the high-content imaging analyzer (CellVoyager CV8000) was directly used for observation; Machine operation: select Cellvis P96-1.5H-N well plate; parameter setting: 10x objective, select 4 regions (10% overlap), collect bright field, non-phase difference mode, select FITC, DAPI, PI channels for layer scanning, 6 layers, 12 μm per layer; Results: Representative images are shown in FIG. 1C. Figure 10 and Figure 11 As shown in FIG. 1C, the organoid drug responsiveness was analyzed using the built-in image analysis software of the high-content imaging analyzer, and two indicators of organoid average area and organoid count were counted.

[0080] As shown in FIG. 1C, the organoid drug responsiveness was analyzed using the built-in image analysis software of the high-content imaging analyzer, and two indicators of organoid average area and organoid count were counted. Figure 12 As shown in FIG. 1C, the organoid drug responsiveness was analyzed using the built-in image analysis software of the high-content imaging analyzer, and two indicators of organoid average area and organoid count were counted.

[0081] Example 9 PDXO and drug responsiveness detection constructed by 3D agarose microarray co-culture The present embodiment provides a DLBCL PXDO operation and characterization method using agarose microarray culture.

[0082] 1. Agarose microarray preparation Microarray hole plate: ultra-thin glass bottom 96-well plate (Cellvis P96-1.5H-N); The OCM formula is the same as described in Example 2; The raw materials, consumables and instruments required for the preparation of the agarose microarray include agarose powder, sterile PBS, autoclaved glass bottles, 6x10 microarray making molds, 96-well plates, microwave ovens, dry metal baths, and water baths.

[0083] The specific steps are as follows: (1) Analytical balance 0.2 g of dry agarose powder, put into a 50 mL autoclaved glass bottle, add 10 mL of sterile PBS to immerse the agarose powder, and rotate gently in the horizontal direction to dissolve as much powder as possible.

[0084] (2) Transfer the glass bottle to the microwave oven and heat to boiling to completely dissolve the agarose powder. During this process, stop heating every 10 seconds, take the glass bottle out of the microwave oven and rotate to promote agarose dissolution, until it is ensured that there are no small translucent agarose fragments left.

[0085] (3) Autoclave the glass bottle containing completely dissolved agarose solution or gel with the cap in a semi-tight state for 30 min, and after sterilization, tighten the cap and put it into the intercellular space for standby.

[0086] (4) Put the autoclaved glass bottle, 6x10 microarray making molds, and dry metal bath together in the biosafety cabinet and irradiate with ultraviolet light for 30 min.

[0087] (5) Turn on the intercellular water bath to warm the water in the water bath to 80 °C for standby, and turn on the dry metal bath to warm it to 80 °C for standby.

[0088] (6) Put the autoclaved glass bottle into the 80 °C water bath to heat, and melt the agarose gel in the bottle until it is ensured that there are no small translucent agarose fragments left.

[0089] (7) Put the 96-well plate into the dry metal bath, and add the melted hot agarose solution to the wells of the 96-well plate at an amount of 70 μL per well, avoiding the generation of air bubbles when aspirating or pipetting the agarose solution.

[0090] (8) Gently press the prepared mold vertically into the 96-well plate with the agarose solution prepared on the dry metal bath.

[0091] (9) Adjust the temperature of the dry metal bath equipment to 20 °C, and cool the agarose solution in the 96-well plate with the pressed-in mold for 3-5 min to completely solidify the agarose solution.

[0092] (10) After the agarose is solidified, transfer the 96-well plate on the dry metal bath and place it flat on the biosafety cabinet operating table, gently pull out the mold vertically, and complete the whole plate preparation of the agarose microwell array.

[0093] 2. Cell preparation (1) MRC5-LB cell preparation MRC5-LB cell recovery and culture: Recover MRC5-LB cells to 2 6 cm sterile culture dishes 3-5 days in advance, and the MRC5-LB cell culture medium formula is the same as described in Example 1, and the culture conditions are: 37 °C, 5% CO2; MRC5-LB cell collection and counting: trypsinize and collect MRC5-LB cells, centrifuge at 250 g for 5 min, discard the supernatant, resuspend with OCM and count; (2) DLBCL PDXC DLBCL PDXC recovery: Take the frozen DLBCL PDXC from liquid nitrogen and place it in a 37 °C water bath to quickly warm it up, centrifuge at 400 g for 5 min after adding 5 mL of complete medium, discard the supernatant, resuspend the cells with OCM and count, prepare about 4E6 PDXC; Prepare a mixed cell suspension: according to the total cell number of 1.5E5 cells / well, 2E6 cells are needed for 12 wells; DLBCL PDXC: MRC5-LB cell = 9:1, i.e. DLBCL PDX cell 1.8E6, MRC5-LB 2E5, prepare a mixed cell suspension with a cell density of 1.5E6 cells / mL with OCM.

[0094] 3. Discard the original complete medium in the cell-seeded hole plate, add 100 μL of mixed cell suspension (containing 1.5E5 cells) to each hole, stand for 15-20 min; observe the cell sedimentation under the microscope, and transfer to a 37°C, 5% CO2 incubator for culture; the next day, observe the cell sphere formation under the microscope, and observe the bright field and FITC channel; 4. Drug responsiveness detection of PDXO Drug solution preparation: select chidamide as the screening drug, and the experimental group drug concentration is 10 μM. Since each hole originally contains 100 μL of OCM, 2x drug solution is prepared, specifically: 1.6 μL of 10 mM chidamide + 798.4 μL of OCM, prepare a drug solution with a final concentration of 20 μM and a total volume of 800 μL; Grouping design: the grouping includes a control group and an experimental group, and 6 replicates are set for each group; Drug treatment: add 100 μL of the prepared 2x drug solution to each hole of the experimental group, and add 100 μL of complete medium to the control group; Drug response observation: take photos of the organoids in the micro-hole plate using a high-content imaging analyzer (Perkin Elmer Operetta) 48 h after drug administration; Staining treatment: the staining solution formula is 1.5 μM PI dye + 20 μM Hoechst-33342 dye + PBS; discard the original culture medium, add 50 μL of staining solution to each hole, and directly perform subsequent shooting on the machine; Machine operation: hole plate selection: Cellvis P96-1.5H-N; magnification: 2x lens; environment setting: 37°C, 5% CO2; channel setting: bright field, FITC, Hoechst, PI; Z stack: 8x20 μm; data processing: ImageJ processes the bright field and fluorescence images taken by the high-content imaging analyzer, and exports the total fluorescence intensity of each channel of the organoid sphere; the data exported above is analyzed using GraphPad Prism software, and non-paired T test is performed, with P value less than 0.05 defined as significant difference.

[0095] The imaging results are shown in Figure 13 , the bright field photos of the control group and the experimental group are shown in Figure 13 , and the fluorescence merged photos of the control group and the experimental group are shown in Figure 13 , it can be seen that the PI corresponding channel fluorescence intensity and area of the experimental group are obviously stronger than those of the control group.

[0096] The data processing results are shown in Figure 14 , the average fluorescence intensity values of different channels of the control group and the experimental group are respectively counted, non-paired T test is performed, and within the 95% confidence interval,Figure 14 (Left) P = 0.1115, Figure 14 (Middle) P = 0.0010, Figure 14 (Right) P = 0.0098, indicating that the cediranib drug treatment has no significant effect on the growth of MRC5-LB cells, but the cediranib drug treatment significantly inhibits the growth of DLBCL PDXO, leading to significant death of DLBCL cells.

[0097] Example 10 Construction and drug sensitivity detection of agarose microarray cocultured mantle cell lymphoma (MCL) PDO The present embodiment provides an operation and characterization method for drug screening by culturing MCL PDO using agarose microarray.

[0098] 1. Agarose microarray preparation as described in Example 9; 2. Cell preparation (1) MRC5-LB cell preparation: MRC5-LB cell recovery and culture: MRC5-LB cells were recovered 3-5 days in advance into 2 6 cm sterile culture dishes, and the MRC5-LB cell culture medium formula is as described in Example 1, and the culture conditions are: 37°C, 5% CO2; MRC5-LB cell collection and counting: MRC5-LB cells were collected by trypsin digestion, and after centrifugation at 250 g for 5 min, the supernatant was discarded, and the cells were resuspended with OCM and then counted; (2) Mantle cell lymphoma patient-derived cells (MCL PDC): MCL PDC recovery: The frozen MCL PDC was taken out from liquid nitrogen and placed in a 37°C water bath for rapid warming, and after adding 5 mL OCM, it was centrifuged at 400 g for 5 min, and the supernatant was discarded; Cell counting: After resuspending the cells with OCM, count about 4E6 MCL PDC, and the OCM formula is as described in Example 2; Prepare mixed cell suspension: according to the total cell number of 1.5E5 cells / well, 20 wells need to prepare 3E6 cells; MCL PDC: MRC5-LB cells = 9:1, i.e. MCL PDC needs 2.7E6, and MRC5-LB needs 3E5, and prepare a mixed cell suspension with a cell density of 1.5E6 / mL with OCM.

[0099] 3. Cell inoculation and observation Cell inoculation: aspirate the original culture medium in the well plate, add 100 μL of mixed cell suspension (containing 1.5E5 cells) to each well, and stand for 15-20 min; Observation and culture: observe the cell sedimentation under the microscope, and transfer to 37°C, 5% CO2 for culture; Spheroid observation: The next day after inoculation, the spheroid formation of the co-cultured cells in the microplate was observed under a microscope, and the bright field and FITC channels were observed. 4. Drug responsiveness detection of MCL PDO Drug solution preparation: Venetoclax was selected as the screening drug, and the drug concentrations of the administration groups were 10 nM, 100 nM, 1 μM, and 10 μM. Since each well originally contained 100 μL OCM, 2x drug solutions were prepared, specifically: 2 μL 10 mM Venetoclax + 498 μL OCM, to prepare a drug solution with a final concentration of 20 μM and a total volume of 500 μL; 50 μL 20 μM Venetoclax + 450 μL OCM, to prepare a drug solution with a final concentration of 2 μM and a total volume of 500 μL; 50 μL 2 μM Venetoclax + 450 μL OCM, to prepare a drug solution with a final concentration of 200 nM and a total volume of 500 μL; and 50 μL 200 nM Venetoclax + 450 μL OCM, to prepare a drug solution with a final concentration of 20 nM and a total volume of 500 μL. Grouping design: The groups included a control group and experimental groups (10 nM, 100 nM, 1 μM, and 10 μM), and each group had 3 replicates. Drug administration: 100 μL of the aforementioned 2x drug solution was added to each well of the experimental groups, and 100 μL of OCM was added to the control group. Drug response observation: After 72 h of drug administration, the organoids in the microplate were photographed using a high-content imaging analyzer (Perkin Elmer Operetta). Staining treatment: The staining solution was prepared with 1.5 μM PI dye + 20 μM Hoechst-33342 dye + PBS. The original culture medium was aspirated, and 50 μL of the staining solution was added to each well for subsequent imaging. Machine operation: Plate selection: Cellvis P96-1.5H-N; magnification: 10x objective; environmental settings: 37°C, 5% CO2; channel settings: bright field, FITC, Hoechst-33342, PI; Z stack: 20x20 μm; data processing: the bright field and fluorescence images photographed by the high-content imaging analyzer were processed using ImageJ software, and the total fluorescence intensity of each channel of the organoid sphere was exported; the data exported above were analyzed using GraphPad Prism software, one-way ANOVA was performed, and P values less than 0.05 were defined as significant differences.

[0100] The imaging results are as follows Figure 15As shown, the fluorescence of the control group and the administration group was merged, and it could be seen that the fluorescence intensity and area of PI corresponding channel of the venetoclax 1 μM and 10 μM groups were obviously stronger than those of the control group.

[0101] The data processing results are shown in Figure 16 As shown, the ratio of the total fluorescence intensity of PI to the total fluorescence intensity of Hoechst of the control group and the administration group was respectively calculated, and one-way ANOVA was performed, and within the 95% confidence interval, the venetoclax 1 μM group P = 0.0162) and the venetoclax 10 μM group The ratio of the total fluorescence intensity of PI to the total fluorescence intensity of Hoechst of the control group was significantly higher than that of the control group, which indicated that the high-dose venetoclax drug treatment significantly inhibited the growth of MCL PDO, resulting in the significant death of MCL PDO.

[0102] The MCL PDO (control group) was subjected to histological identification, and the specific operation was the same as that in Example 6 (only H&E staining was performed in this example), and the representative results are shown in Figure 17 As shown, the central part of the MCL PDO was MRC5-LB, and the surrounding was MCL PDC, and there was cell-to-cell crosstalk between the MCL PDC and the MRC5-LB cells, forming a dense organoid ball.

[0103] Although the present application has been described in detail in the foregoing embodiment, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application are within the scope of the present application.

[0104] References: [1] ENNISHI D, HSI E D, STEIDL C, et al. Toward a new molecular taxonomy of diffuse large B-cell lymphoma[J]. Cancer Discovery, 10(9): 1267-1281. [2] ARBER D A, ORAZI A, HASSERJIAN R, et al. The 2016 revision to the world health organization classification of myeloid neoplasms and acute leukemia[J]. Blood, 127(20): 2391-2405. [3] GUO R, WANG W, YU L, et al. Different regulatory effects of CD40 ligand and B-cell activating factor on the function of B cells[J]. International Immunopharmacology, 91: 107337. [4] WEEBER F, OOFT S N, DIJKSTRA K K, et al. Tumor organoids as a pre-clinical cancer model for drug discovery[J]. Cell Chemical Biology, 24(9): 1092-1100. [5] SHAH S B, CARLSON C R, LAI K, et al. Combinatorial treatment rescues tumor-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas[J]. Nature Materials, 22(4): 511-523. [6] ROSSI M, ALVIANO F, RIGHI S, et al. Three-dimensional models: A novel approach for lymphoma research[J]. Journal of Cancer Research and Clinical Oncology, 148(4): 753-765. [7] YANGUAS-CASÁS N, PEDROSA L, FERNÁNDEZ-MIRANDA I, et al. An overview on diffuse large B-cell lymphoma models: Towards a functional genomics approach[J]. Cancers, 13(12): 2893.

Claims

1. A method of culturing lymphoma organoids PDO, characterized by, The method comprises: co-culturing lymphoma cells with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF in vitro; The lymphoma cells are B-cell lymphoma cells, and are derived from patient-derived xenograft cells PDXC or patient-derived primary tumor cells PDC; The engineered stromal cells ESC are fibroblasts or stromal cells overexpressing CD40L, BAFF and / or other cell factors promoting growth of lymphoma, and are derived from mice or humans.

2. The method of claim 1, wherein, The construction method of the engineered stromal cells ESC comprises: constructing a lentivirus vector, stably transfecting CD40L, BAFF and / or other cell factors promoting growth of lymphoma into fibroblasts to obtain the engineered stromal cells ESC.

3. The method of claim 1, wherein, The preparation method of the patient-derived tumor-associated fibroblasts CAF and the patient-derived primary tumor cells PDC comprises: taking a lymph tissue sample of a B-cell lymphoma patient, cutting the sample after washing, treating the sample with a digestive enzyme, discarding the supernatant after centrifugation, and resuspending the cells with a human tissue washing solution; then, the patient-derived tumor-associated fibroblasts CAF and the patient-derived primary tumor cells PDC are enriched, amplified and separated by using the characteristics that fibroblasts adhere to a substrate and lymphoma cells grow in suspension.

4. The method of claim 3, wherein, The formula of the digestive enzyme is: 0.1 mg / mL DNase + 1 mg / mL collagenase IV + 10 μM Y-27632, and the human tissue washing solution is prepared; The formula of the human tissue washing solution is: RPMI 1640 medium + 2 mM GlutaMAX supplement + 10 mM HEPES + 100 μg / mL Primocin + 1% BSA.

5. The method of claim 3, wherein, The patient-derived tumor-associated fibroblasts CAF are cultured in a patient-derived tumor-associated fibroblast CAF culture medium; the formula of the patient-derived tumor-associated fibroblast CAF culture medium is: DMEM / F12 + 10% FBS + 1% penicillin-streptomycin + 10 ng / mL bFGF + 10 mM HEPES + 2 mM GlutaMAX supplement + 1 mM sodium pyruvate + 0.1 mM non-essential amino acids; The patient-derived primary tumor cells PDC are stored in a primary cryopreservation solution; the formula of the primary cryopreservation solution is: DMEM + 1% penicillin-streptomycin + 2 mM GlutaMAX supplement + 50% FBS + 10% DMSO + 10 μM Y-27632.

6. The method of claim 1, wherein, The lymphoma cells are mixed with the engineered stromal cells ESC or the patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1 to 1:1, inoculated in a lymphoma organoid culture medium, and co-cultured in vitro. The lymphoma organoid culture medium is formulated as follows: DMEM / F12 + 10% FBS + 1% penicillin-streptomycin + 10 mM HEPES + 2 mM GlutaMAX supplement + 10 ng / mL IL-4.

7. The method of claim 1, wherein, The in vitro co-culture is performed in any of the following ways: (a) 2.5D co-culture: lymphoma cells are seeded on engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1 in a lymphoma organoid culture medium for 2.5D culture; (b) 3D ultra-low attachment culture: lymphoma cells are mixed with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1, seeded in a 96-well flat-bottom ultra-low attachment multi-well plate, and cultured in suspension using a lymphoma organoid culture medium; (c) 3D Matrigel co-culture: lymphoma cells are mixed with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1, and the resulting cell suspension is embedded with Matrigel at a volume ratio of 1:1 to form a three-dimensional structure for culture; (d) 3D agarose microarray co-culture: lymphoma cells are mixed with engineered stromal cells ESC or patient-derived tumor-associated fibroblasts CAF at a ratio of 20:1-1:1 in a lymphoma organoid culture medium, seeded into a pre-formed agarose microwell array, and cultured after standing for 10-30 min; (e) other commercial microwell plates that can be used for 50 μm~500 μm microsphere culture; The lymphoma organoid culture medium is as described in claim 6.

8. The method of claim 7, wherein, The in vitro co-culture is performed in 2.5D or 3D.

9. Use of the lymphoma organoid PDO obtained by the method of any one of claims 1-8 in anti-tumor drug screening.

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