A prostate cancer drug evaluation organoid chip and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine and tumor drug evaluation, and particularly relates to a prostate cancer drug evaluation organoid chip and a preparation method and application thereof. The organoid chip is filled with co-culture freeze-dried reagents in the co-culture hole of the co-culture area, and is designed in cooperation with an oxygen concentration gradient, so that the prostate cancer tumor-immune microenvironment can be simulated. The application provides a precise platform for prostate cancer ADC drug screening and efficacy prediction, and is suitable for preparation of a prostate cancer drug efficacy evaluation product and actual efficacy evaluation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine and tumor drug evaluation, and particularly relates to a prostate cancer drug evaluation organoid chip and a preparation method and application thereof. BACKGROUND

[0002] Immunotherapy has become an important breakthrough direction in the field of prostate cancer treatment by activating the body's adaptive immune system to target tumors, especially in inhibiting tumor metastasis and recurrence. However, the immune microenvironment of prostate cancer tissue has significant immunosuppressive characteristics. This characteristic is not simply a decrease in the number of immune cells, but rather a deep "deactivation" of immune cell function. The key effector cell CD8+ T cell has a significantly reduced expression level of surface activation markers (such as CD28 and CD69) and impaired cytokine (such as IFN-γ and TNF-α) secretion capacity, exhibiting a phenotype similar to that of an unactivated initial T cell, which prevents it from differentiating into a CTL (cytotoxic effector CD8+ T cell) with high anti-tumor activity. This not only restricts the efficacy of simple immunotherapy, but also poses a severe challenge to ADC drugs that rely on targeted killing and immune synergy.

[0003] Antibody drug conjugate (ADC) drugs are new anti-tumor drugs with precise targeting and high killing efficiency. Their core advantage is the ability to precisely deliver highly toxic cytotoxic drugs to tumor cells through the specific targeting ability of antibodies, reducing damage to normal tissues while improving killing efficiency. However, in the context of prostate cancer treatment, the efficacy of ADC drugs faces double obstacles. On the one hand, the expression of some target antigens on the surface of prostate cancer cells is heterogeneous, which may cause some tumor cells to escape ADC drug targeting recognition. On the other hand, even if ADC drugs kill some tumor cells, the released tumor antigens need to be captured by antigen-presenting cells and activate CTL anti-tumor immune responses to achieve the cycle of "killing-activation-killing". The immunosuppressive microenvironment of prostate cancer inhibits this process: for example, regulatory T cells in the tumor microenvironment secrete IL-10, TGF-β, and other inhibitory cytokines to inhibit the activation and proliferation of CTLs; myeloid suppressor cells weaken the killing function of CTLs by consuming amino acids in the microenvironment and producing reactive oxygen species. Therefore, the efficacy of ADC drugs in prostate cancer not only depends on their direct killing ability of tumor cells, but also depends on their ability to break through the immunosuppressive microenvironment and form a synergistic effect with the body's immune cells.

[0004] Traditional ADC drug evaluation models are difficult to comprehensively simulate the above-mentioned complex tumor-immune interaction process, resulting in low drug clinical conversion efficiency. Specifically, although the traditional cell line model can be used to evaluate the direct killing effect of ADC drugs, these cell lines have lost the antigen heterogeneity and microenvironment adaptability of primary tumor tissues after long-term in vitro culture, and cannot simulate the phenotypic characteristics of tumor cells in vivo. At the same time, the cell line culture system lacks immune cells and cannot evaluate the synergistic effect of ADC drugs and immune cells. Although the animal model can simulate the tumor growth process to a certain extent, the immune deficiency state cannot restore the immunosuppressive microenvironment of human prostate cancer; while the humanized mouse model can construct a human immune system, but its construction cost is high, the cycle is long, and there are species differences in the functions of immune cells and the composition of tumor microenvironment between mice and humans, making it difficult to accurately predict the efficacy and side effects of ADC drugs in human patients.

[0005] Although the tumor organoid can retain the antigen specificity and heterogeneity of tumor tissue, the traditional organoid culture system lacks the spatial structure and dynamic environment of immune cell co-culture, and cannot simulate the in vivo tumor-immune cell interaction process, making it difficult to accurately evaluate the direct killing effect of ADC drugs on tumor cells and the regulatory effect on immune cells (such as CTL).

[0006] Therefore, it is urgent to construct an organoid chip model that can simulate the prostate cancer tumor-immune microenvironment and achieve multi-dimensional evaluation of ADC drugs. SUMMARY

[0007] In view of the above problems, the present application provides an organoid chip for prostate cancer drug evaluation and a preparation method and application thereof.

[0008] The technical scheme of the present application is as follows:

[0009] On the one hand, the present application provides an organoid chip for prostate cancer drug evaluation, which comprises a chip body, wherein the chip body comprises a co-culture area, the co-culture area is provided with a co-culture well, and the co-culture well is filled with co-culture freeze-dried reagents.

[0010] The co-culture freeze-dried reagents are obtained by freeze-drying of co-culture reagents, and the co-culture reagents consist of IL-2, IL-15, anti-PD-L1 antibody, deferoxamine, HEPES, FGF-2, trehalose, mannitol, BSA and culture medium.

[0011] The use method of the organoid chip comprises the following steps:

[0012] S1, adding the organoid suspension and the PBMC suspension into the co-culture well, and performing primary co-culture under 10%-12% oxygen;

[0013] S2, after the initial co-culture is finished, the initial culture solution is discarded, and the organoid suspension is added again, and the secondary co-culture is carried out under 15%-18% oxygen to obtain a secondary co-culture product;

[0014] S3, after the secondary co-culture product is co-cultured with the prostate cancer organoids, the ADC drug to be tested is added, and after culture, it is used for efficacy evaluation.

[0015] Specifically, the co-culture reagent comprises 15-20 IU / mL IL-2, 10-15 ng / mL IL-15, 3.5-5.0 μg / mL anti-PD-L1 antibody, 50-60 μmol / L desferrioxamine, 10-15 mmol / L HEPES, 5-8 ng / mL FGF-2, 2%-3% w / v trehalose, 2%-5% w / v mannitol and 0.1%-0.5% w / v BSA.

[0016] Further specifically, the co-culture reagent comprises 15-16, 16-17, 17-18, 18-19 or 19-20 IU / mL IL-2.

[0017] Preferably, the co-culture reagent comprises 20 IU / mL IL-2.

[0018] Further specifically, the co-culture reagent comprises 10-11, 11-12, 12-13, 13-14 or 14-15 ng / mL IL-15.

[0019] Preferably, the co-culture reagent comprises 15 ng / mL IL-15.

[0020] Further specifically, the co-culture reagent comprises 3.5-3.6, 3.6-3.7, 3.7-3.8, 3.8-3.9, 3.9-4.0, 4.0-4.1, 4.1-4.2, 4.2-4.3, 4.3-4.4, 4.4-4.5, 4.5-4.6, 4.6-4.7, 4.7-4.8, 4.8-4.9 or 4.9-5.0 μg / mL anti-PD-L1 antibody.

[0021] Preferably, the co-culture reagent comprises 5.0 μg / mL anti-PD-L1 antibody.

[0022] Further specifically, the co-culture reagent comprises 50-51, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59 or 59-60 μmol / L desferrioxamine.

[0023] Preferably, the co-culture reagent comprises 50 μmol / L desferrioxamine.

[0024] Further specifically, the co-culture reagent comprises 10-11, 11-12, 12-13, 13-14, or 14-15 mmol / L HEPES.

[0025] Preferably, the co-culture reagent comprises 10 mmol / L HEPES.

[0026] Further specifically, the co-culture reagent comprises 5-6, 6-7, or 7-8 ng / mL FGF-2.

[0027] Preferably, the co-culture reagent comprises 5 ng / mL FGF-2.

[0028] Further specifically, the co-culture reagent comprises 2.0%-2.1%, 2.1%-2.2%, 2.2%-2.3%, 2.3%-2.4%, 2.4%-2.5%, 2.5%-2.6%, 2.6%-2.7%, 2.7%-2.8%, 2.8%-2.9%, or 2.9%-3.0% w / v trehalose.

[0029] Preferably, the co-culture reagent comprises 2.0% w / v trehalose.

[0030] Further specifically, the co-culture reagent comprises 2.0%-2.1%, 2.1%-2.2%, 2.2%-2.3%, 2.3%-2.4%, 2.4%-2.5%, 2.5%-2.6%, 2.6%-2.7%, 2.7%-2.8%, 2.8%-2.9%, 2.9%-3.0%, 3.0%-3.1%, 3.1%-3.2%, 3.2%-3.3%, 3.3%-3.4%, 3.4%-3.5%, 3.5%-3.6%, 3.6%-3.7%, 3.7%-3.8%, 3.8%-3.9%, 3.9%-4.0, 4.0%-4.1%, 4.1%-4.2%, 4.2%-4.3%, 4.3%-4.4%, 4.4%-4.5%, 4.5%-4.6%, 4.6%-4.7%, 4.7%-4.8%, 4.8%-4.9%, or 4.9%-5.0% w / v mannitol.

[0031] Preferably, the co-culture reagent comprises 2.0% w / v mannitol.

[0032] Further specifically, the co-culture reagent comprises 0.1%-0.2%, 0.2%-0.3%, 0.3%-0.4%, or 0.4%-0.5% w / v BSA.

[0033] Preferably, the co-culture reagent comprises 0.1% w / v BSA.

[0034] Specifically, the organoid suspension in step S1 or step S2 is added according to the ratio of the number of cells of the organoids in the organoid suspension to the number of cells of the PBMCs in the PBMC suspension; the ratio of the number of cells of the organoids in the organoid suspension to the number of cells of the PBMCs in the PBMC suspension is 1:15-25.

[0035] Preferably, the organoid suspension in step S1 or step S2 is added according to the ratio of the number of cells of the organoids in the organoid suspension to the number of cells of the PBMCs in the PBMC suspension; the ratio of the number of cells of the organoids in the organoid suspension to the number of cells of the PBMCs in the PBMC suspension is 1:20.

[0036] Specifically, the prostate cancer organoid in step S3 is added according to the ratio of the number of cells of the prostate cancer organoid to the number of cells of the PBMCs in the PBMC suspension; the ratio of the number of cells of the prostate cancer organoid to the number of cells of the PBMCs in the PBMC suspension is 1:10-15.

[0037] Preferably, the prostate cancer organoid in step S3 is added according to the ratio of the number of cells of the prostate cancer organoid to the number of cells of the PBMCs in the PBMC suspension; the ratio of the number of cells of the prostate cancer organoid to the number of cells of the PBMCs in the PBMC suspension is 1:10.

[0038] Specifically, the conditions of the primary co-culture in step S1 are 35-40℃, 5% CO2, 10%-12% oxygen, and the culture is performed for 7-9 days.

[0039] Preferably, the conditions of the primary co-culture in step S1 are 37℃, 5% CO2, 10% oxygen, and the culture is performed for 7 days.

[0040] Specifically, the conditions of the secondary co-culture in step S2 are 35-40℃, 5% CO2, 15%-18% oxygen, and the culture is performed for 5-7 days.

[0041] Preferably, the conditions of the secondary co-culture in step S2 are 37℃, 5% CO2, 18% oxygen, and the culture is performed for 5-7 days.

[0042] Specifically, the conditions of the co-culture in step S3 are 35-40℃, 5% CO2 for 2-5h; and the culture conditions are 35-40℃, 5% CO2 for 48-72h.

[0043] Preferably, the conditions of the co-culture in step S3 are 37℃, 5% CO2 for 2h; and the culture conditions are 37℃, 5% CO2 for 48h.

[0044] Specifically, the organoid chip further comprises a cell loading area, a sample loading area and a waste collection area, and each area is communicated through a microfluidic channel.

[0045] In another aspect, the application provides a preparation method of the organoid chip, comprising the following steps:

[0046] (1) adding co-culture reagent into the co-culture well, freeze-drying, and covering the co-culture area with a sterile sealing film;

[0047] (2) sequentially assembling the cell loading area, the co-culture area, the sample loading area and the waste collection area, and each area is communicated through a microfluidic channel;

[0048] (3) sterilizing after assembly, connecting an oxygen sensor and a temperature sensor, and obtaining the organoid chip.

[0049] In another aspect, the application provides an application of the organoid chip, comprising any one or more of the following:

[0050] (1) an application in the preparation of a prostate cancer drug efficacy evaluation product;

[0051] (2) an application in the efficacy evaluation of a prostate cancer drug.

[0052] In another aspect, the application provides a prostate cancer drug efficacy evaluation product, which comprises the organoid chip.

[0053] In another aspect, the application provides a method for evaluating the efficacy of a prostate cancer drug, which comprises using the organoid chip or the efficacy evaluation product.

[0054] The application has the following advantages:

[0055] The application provides a precise platform for screening and efficacy prediction of prostate cancer ADC drugs, and is suitable for the preparation and actual efficacy evaluation of a prostate cancer drug efficacy evaluation product. DETAILED DESCRIPTION

[0056] The application will be further clarified by the following examples, which are only a part of the examples of the application and are not used to limit the application. The experimental methods used in the following examples are conventional experiments, and the materials and reagents used in the following examples, such as anti-PD-L1 antibody (purchased from R&D Systems), are commercially available unless otherwise specified.

[0057] The organoid chip of the present invention includes a chip body, which sequentially comprises a cell loading area, a co-culture area, a sample loading area, and a waste collection area, all connected by microchannels. The cell loading area includes organoid suspension loading wells, PBMC suspension loading wells, and PDO cell loading wells. The bottom of each loading well is connected to the co-culture area via a microchannel, enabling the delivery of organoid suspension, PBMC suspension, or PDO cells. The co-culture area includes co-culture wells filled with co-culture lyophilized reagents; the co-culture area also includes an oxygen concentration monitoring interface and a temperature sensor interface for real-time monitoring of culture environment parameters. The sample loading area is located between the co-culture area and the waste collection area, and is connected to the co-culture area one-to-one via an array of microchannels. The sample loading area has multiple sets of independent drug loading wells, each set containing three parallel loading wells; each set of drug loading wells is connected to the corresponding three co-culture wells in the co-culture area via three independent microchannels. The waste liquid collection area is provided with one main waste liquid collection chamber and multiple branch waste liquid chambers. The branch waste liquid chambers are connected to the co-culture wells of the corresponding group through independent microchannels. The microchannels have a slope of 5° and utilize gravity to assist the gravity flow of waste liquid. The main collection chamber and the branch collection chambers are connected by a one-way valve to prevent waste liquid from flowing back and contaminating the co-culture area.

[0058] Basic Example 1: Construction and Identification of Prostate Cancer Organoids

[0059] Advanced DMEM / F12 was used as the basal culture medium, and recombinant human epidermal growth factor 50 ng / mL, recombinant human Noggin 100 ng / mL, recombinant human R-spondin 1 1000 ng / mL, recombinant human FGF-10 500 ng / mL, recombinant human FGF-basic 20 ng / mL, and A83-01 5 μM were added to obtain prostate cancer culture medium.

[0060] Fresh tumor tissue samples were collected from the patient after surgical removal and washed four times with sterile PBS buffer, 5 minutes each time. The washed tissue was then cut into pieces approximately 1 mm in size using a scalpel. 3 Transfer tissue samples of this size to 15 mL centrifuge tubes and add an appropriate amount of TrypLE. TM The Express enzyme was placed in a 37°C water bath shaker at 60 rpm for 40 minutes, gently shaking every 5 minutes. After digestion, the digestion process could be observed under a microscope. After digestion was stopped, the sample was pipetted and filtered through a 100-mesh filter to obtain a cell clump suspension. Finally, the suspension was centrifuged at 1500 rpm for 10 minutes in a pre-cooled 4°C centrifuge, and the supernatant was discarded. This process was repeated three times to obtain the cell clump pellet.

[0061] Then, the cell cluster precipitate was mixed with Matrigel. ®The matrix was mixed at a volume ratio of 1:1 to obtain a cell mass, which was added to the prostate cancer culture medium for organoid construction at 37°C and 5% CO2.

[0062] By the fourth day, obvious PCa PDO spheres were visible in the field of view, and the edges were clear, indicating that they were in good condition. Continued expansion to D9 days showed that the organoids continued to grow, with an average diameter of about 100 pm, and continued to be passaged for expansion. By the fifth day of T1 generation, some spheres had reached about 200 pm and were in good growth condition and could be used for subsequent experiments, referred to as prostate cancer organoids (PDO).

[0063] The above prostate cancer organoids were collected and subjected to HE and Epcam staining, and compared with the tissue. The PDO presented a multi-layered cell structure, with a stromal component in the lumen, and a distribution of nuclei and cytoplasm consistent with the tissue structure; Epcam is an epithelial-specific marker that is increased in tumor tissue, and the positive proportion and fluorescence intensity of PDO staining are similar to those of the tissue. This indicates the consistency of PDO with the morphology of the source tissue at the morphological level.

[0064] Example 2: Recovery of PBMCs from healthy humans

[0065] The PBMC cryopreservation tube was placed in a 37°C water bath, gently shaken until completely melted (2 min), 18 mL of IMDM complete medium was added to the centrifuge tube, the PBMCs were added, centrifuged at 500g for 5 min, the supernatant was discarded, the cell mass was loosened by tapping, and 20 mL of IMDM complete medium was added, which was gently blown 3-5 times to prepare a PBMC cell suspension.

[0066] Example 1: A prostate cancer drug evaluation organoid chip

[0067] 1. Preparation of organoid chip

[0068] 1.1 Preparation of co-culture reagent

[0069] The co-culture reagent is DMEM / F12 medium containing 20 IU / mL IL-2, 15 ng / mL IL-15, 5 pg / mL anti-PD-L1 antibody, 50 pmol / L desferrioxamine, 10 mmol / L HEPES, 5 ng / mL FGF-2, 2% w / v trehalose, 2% w / v mannitol, and 0.1% w / v BSA.

[0070] The preparation method of the co-culture reagent is as follows: add each component to the DMEM / F12 medium in the above-mentioned final concentration, filter sterilize with a 0.22 pm filter membrane, and obtain the co-culture reagent.

[0071] 1.2 Filling and freeze-drying of co-culture reagent

[0072] Put the co-culture well of the chip upwards, and add 50 μL of co-culture reagent; place the chip in an ultra-low temperature freezer at -80°C for pre-freezing for 3 h, and then transfer it to a vacuum freeze dryer (vacuum degree: 0.08 mbar, temperature: -55°C) for freeze-drying for 15 h until the reagent is completely in the form of loose white powder; immediately after freeze-drying is completed, cover the co-culture area with a sterile sealing film.

[0073] 1.3 Assembly and sterilization of organ chip

[0074] The organ chip comprises a chip body, which is sequentially provided with a cell loading area, a co-culture area, a sample loading area, and a waste liquid collection area, and each area is connected through a microchannel. After assembly is completed, the chip is placed in a UV sterilization box (wavelength: 254 nm) for irradiation for 30 min, and then connected to an oxygen sensor (detection range: 0-25%) and a temperature sensor (detection range: 30-40°C) for standby.

[0075] 1.4 Chip loading and co-culture process

[0076] S1, organ suspension loading: take the prostate cancer organoids prepared in basic example 1, wash twice with PBS, adjust the concentration to 1×10 5 / mL to obtain a PDO suspension, and inject 25 μL of the PDO suspension (2.5×10 3 PDOs per well) into the co-culture well through the organ suspension loading hole;

[0077] S2, PBMC suspension loading: take the PBMC suspension (2×10 6 / mL) prepared in basic example 2, inject 25 μL of the suspension (5×10 4 cells per well) into the co-culture well through the PBMC suspension loading hole, so that the number ratio of organoids to PBMCs is 1:20;

[0078] S3, primary co-culture: place the chip in a culture box at 37°C, 5% CO2, and an oxygen concentration of 10%, and co-culture for 7 days;

[0079] S4, secondary co-culture: on the 7th day (D7) of co-culture, slowly remove 50% of the old culture solution in the co-culture well through the microchannel, inject 25 μL of the PDO suspension (2.5×10 3 PDOs per well) into the co-culture well through the organ suspension loading hole, and co-culture for 5 days (D12) at 37°C, 5% CO2, and an oxygen concentration of 18%.

[0080] 2, use of organ chip

[0081] (1) PDO cell loading: Take the prostate cancer organoids in the basic example 1, wash twice with sterile PBS buffer, centrifuge at 1000 rpm for 5 min, discard the supernatant; adjust the concentration of the organoids to 2x10 5 6 / mL with prostate cancer medium, and obtain PDO cell solution; add PDO suspension (5x10 3 6 PDO / well) in an amount of 25 μL per well, and place it in a 37°C, 5% CO2 incubator for 2 h;

[0082] (2) Drug loading: Add 5 μL of drug solution per well to the co-culture well through the sample loading area, and after the drug loading is completed, place the chip back in a 37°C, 5% CO2 incubator and continue to culture for 48 h for efficacy evaluation.

[0083] Example 2: An organoid chip for prostate cancer drug evaluation

[0084] Example 2 differs from example 1 only in that "1.1 Preparation of co-culture reagent" and "1.4 Chip loading and co-culture process" are different. The specific process is as follows:

[0085] 1.1 Preparation of co-culture reagent

[0086] The co-culture reagent is: DMEM / F12 medium containing 15 IU / mL IL-2, 10 ng / mL IL-15, 3.5 μg / mL anti-PD-L1 antibody, 60 μmol / L deferoxamine, 15 mmol / L HEPES, 8 ng / mL FGF-2, 3% w / v trehalose, 5% w / v mannitol, 0.5% w / v BSA.

[0087] The preparation method of the co-culture reagent is: add each component to the DMEM / F12 medium in the above-mentioned final concentration in turn, filter sterilize with a 0.22 μm filter membrane, and obtain the co-culture reagent.

[0088] 1.4 Chip loading and co-culture process

[0089] S1, organoid suspension loading: Take the prostate cancer organoids prepared in the basic example 1, wash twice with PBS, adjust the concentration to 1x10 5 6 / mL, and obtain PDO suspension, inject 25 μL of PDO suspension (2.5x10 3 6 PDO / well) into the co-culture well through the organoid suspension loading well;

[0090] S2, PBMC suspension loading: Take the PBMC suspension (2x10 6 6 / mL) prepared in the basic example 2, inject 25 μL of suspension (5x10 4(2.5x10

[0091] S3, primary co-culture: the chip was placed in a 37℃, 5% CO2, oxygen concentration 12% incubator for 7 days of co-culture;

[0092] S4, secondary co-culture: on the 7th day of co-culture (D7), 50% of the old culture solution in the co-culture well was removed slowly through the micro-channel, and 25 μL of PDO suspension (2.5x10 3 PDO / well) was injected into the co-culture well through the suspension loading well, and co-cultured for 5 days (D12) at 37℃, 5% CO2, and oxygen concentration 15%.

[0093] Comparative Example 1: An organoid chip for evaluating prostate cancer drugs

[0094] The difference between Comparative Example 1 and Example 1 is only that the "1.1 preparation of co-culture reagent" is different. The specific process is as follows:

[0095] 1.1 Preparation of co-culture reagent

[0096] The co-culture reagent is DMEM / F12 medium containing 20 IU / mL IL-2, 15 ng / mL IL-15, 5 μg / mL anti-PD-L1 antibody, 10 mmol / L HEPES, 5 ng / mL FGF-2, 2% w / v trehalose, 2% w / v mannitol, 0.1% w / v BSA.

[0097] The preparation method of the co-culture reagent is: add each component to the DMEM / F12 medium in the above-mentioned final concentration in turn, filter sterilization with a 0.22 μm filter membrane, and obtain the co-culture reagent.

[0098] Comparative Example 2: An organoid chip for evaluating prostate cancer drugs

[0099] The difference between Comparative Example 2 and Example 1 is only that the "1.1 preparation of co-culture reagent" is different. The specific process is as follows:

[0100] 1.1 Preparation of co-culture reagent

[0101] The co-culture reagent is DMEM / F12 medium containing 20 IU / mL IL-2, 5 μg / mL anti-PD-L1 antibody, 50 μmol / L desferrioxamine, 10 mmol / L HEPES, 5 ng / mL FGF-2, 2% w / v trehalose, 2% w / v mannitol, 0.1% w / v BSA.

[0102] The preparation method of the co-culture reagent is as follows: sequentially adding each component to the DMEM / F12 culture medium according to the final concentration, and filtering bacteria with a 0.22 μm filter membrane to obtain the co-culture reagent.

[0103] Comparative Example 3: An organoid chip for evaluating a prostate cancer drug

[0104] Comparative Example 3 differs from Example 1 only in that the “1.1 Preparation of co-culture reagent” is different. The specific process is as follows:

[0105] 1.1 Preparation of co-culture reagent

[0106] The co-culture reagent is: DMEM / F12 culture medium containing 20 IU / mL IL-2, 5 μg / mL anti-PD-L1 antibody, 10 mmol / L HEPES, 2% w / v trehalose, 2% w / v mannitol, and 0.1% w / v BSA.

[0107] The preparation method of the co-culture reagent is as follows: sequentially adding each component to the DMEM / F12 culture medium according to the final concentration, and filtering bacteria with a 0.22 μm filter membrane to obtain the co-culture reagent.

[0108] Comparative Example 4: An organoid chip for evaluating a prostate cancer drug

[0109] Comparative Example 4 differs from Example 1 only in that the “1.4 Chip loading and co-culture process” is different. The specific process is as follows:

[0110] 1.4 Chip loading and co-culture process

[0111] S1, organoid suspension loading: taking the prostate cancer organoids prepared in the base Example 1, washing twice with PBS, adjusting the concentration to 1×10 5 cells / mL to obtain a PDO suspension, and injecting 25 μL of the PDO suspension (2.5×10 3 cells per well) into the co-culture well through the organoid suspension loading hole;

[0112] S2, PBMC suspension loading: taking the PBMC suspension (2×10 6 cells / mL) prepared in the base Example 2, injecting 25 μL of the suspension (5×10 4 cells per well) into the co-culture well through the PBMC suspension loading hole, so that the number ratio of organoids to PBMCs is 1:20;

[0113] S3, primary co-culture: placing the chip in a culture box at 37°C, 5% CO2, and an oxygen concentration of 8%, and co-culturing for 7 days;

[0114] S4, Secondary co-culture: On day 7 of co-culture (D7), 50% of the old culture medium in the co-culture well was removed slowly through the microfluidic channel, 25 μL of PDO suspension (2.5 x 10 3 cells / well) was injected into the co-culture well through the organoid suspension loading well, and co-cultured for 5 days (D12) at 37 °C, 5% CO2, and an oxygen concentration of 20%.

[0115] Comparative Example 5: An organoid chip for prostate cancer drug evaluation

[0116] Comparative Example 5 differs from Example 1 only in the "1.4 Chip loading and co-culture procedure". The specific process is as follows:

[0117] 1.4 Chip loading and co-culture procedure

[0118] S1, Organoid suspension loading: Take the prostate cancer organoids prepared in Basic Example 1, wash twice with PBS, adjust the concentration to 1 x 10 5 cells / mL to obtain a PDO suspension, and inject 25 μL of PDO suspension (2.5 x 10 3 cells / well) into the co-culture well through the organoid suspension loading well;

[0119] S2, PBMC suspension loading: Take the PBMC suspension prepared in Basic Example 2 (2 x 10 6 cells / mL), inject 25 μL of the suspension (5 x 10 4 cells / well) into the co-culture well through the PBMC suspension loading well, so that the number ratio of organoids to PBMCs is 1:20;

[0120] S3, Primary co-culture: Place the chip in a 37 °C, 5% CO2, and 5% oxygen concentration incubator for 7 days of co-culture;

[0121] S4, Secondary co-culture: On day 7 of co-culture (D7), 50% of the old culture medium in the co-culture well was removed slowly through the microfluidic channel, 25 μL of PDO suspension (2.5 x 10 3 cells / well) was injected into the co-culture well through the organoid suspension loading well, and co-cultured for 5 days (D12) at 37 °C, 5% CO2, and an oxygen concentration of 20%.

[0122] Comparative Example 6: An organoid chip for prostate cancer drug evaluation

[0123] Comparative Example 6 differs from Example 1 only in the "1.1 Preparation of co-culture reagents" and "1.4 Chip loading and co-culture procedure". The specific process is as follows:

[0124] 1.1 Preparation of co-culture reagents

[0125] Co-culture reagent: DMEM / F12 medium containing 20 IU / mL IL-2, 15 ng / mL IL-15, 5 μg / mL anti-PD-L1 antibody, 10 mmol / L HEPES, 5 ng / mL FGF-2, 2% w / v trehalose, 2% w / v mannitol, 0.1% w / v BSA.

[0126] The preparation method of the co-culture reagent is: sequentially adding each component to the DMEM / F12 medium according to the above final concentration, filtering and sterilizing with a 0.22 μm filter membrane to obtain the co-culture reagent.

[0127] 1.4 Chip loading and co-culture process

[0128] S1, organoid suspension loading: taking the prostate cancer organoids prepared in the basic example 1, washing twice with PBS, adjusting the concentration to 1×10 5 cells / mL to obtain a PDO suspension, and injecting 25 μL of the PDO suspension (2.5×10 3 cells / PDO / well) into the co-culture well through the organoid suspension loading hole;

[0129] S2, PBMC suspension loading: taking the PBMC suspension (2×10 6 cells / mL) prepared in the basic example 2, injecting 25 μL of the suspension (5×10 4 cells / well) into the co-culture well through the PBMC suspension loading hole, so that the number ratio of organoids to PBMCs is 1:20;

[0130] S3, primary co-culture: placing the chip in a culture box at 37°C, 5% CO2, and an oxygen concentration of 5%, and co-culturing for 7 days;

[0131] S4, secondary co-culture: on the 7th day (D7) of co-culture, slowly removing 50% of the old culture solution in the co-culture well through the microchannel, injecting 25 μL of the PDO suspension (2.5×10 3 cells / PDO / well) into the co-culture well through the organoid suspension loading hole, and co-culturing for 5 days (D12) at 37°C, 5% CO2, and an oxygen concentration of 12%.

[0132] Experimental example 1: flow cytometry identification of co-culture products

[0133] The cells (step S4 secondary co-culture product) co-cultured in example 1 to example 2 or comparative example 1 to comparative example 6 on the 12th day (D12) were washed twice with PBS (containing 2% FBS), centrifuged at 1500 rpm for 5 min, and the supernatant was discarded; the cells were resuspended in PBS to a concentration of 1×10 6 cells / mL, passed through a 70 μm cell screen, and 100 μL of the cell suspension was added to a flow tube.

[0134] Take 100 μL of cell suspension into a flow tube, add FITC-CD3 antibody (5 μL), PE-CD8 antibody (5 μL), and APC-CD107a antibody (5 μL) respectively, and incubate at 4°C in the dark for 30 min; wash with 2 mL of PBS (containing 2% FBS) at 1500 rpm for 5 min, discard the supernatant; resuspend with 300 μL of PBS (containing 2% FBS), and detect by flow cytometry (BD FACS Canto II), and analyze the data by FlowJo software. The determination results are shown in Table 1:

[0135] Table 1

[0136]

[0137] Experimental Example 2 Killing ability on prostate cancer organoids

[0138] The cells (secondary co-culture product of step S4) of Example 1-Example 2 or Comparative Example 1-Comparative Example 6 are used as effector cells, and the prostate cancer organoids in the basic experimental example 1 are used as target cells. Through the double staining method of CMTPX (prostate cancer organoid red fluorescent label) and CFDA SE (CTL green fluorescent label), combined with the co-stimulation of anti-CD28 coated culture plates, the killing process (prostate cancer organoid quantity reduction) and self-activation and proliferation (CTL quantity increase) of CTL on prostate cancer organoids under the condition of effector-target ratio of 5:1 are observed in real time, and the target killing ability of the cell drug is directly verified.

[0139] 1. Dilute the anti-CD28 antibody to a final concentration of 5 μg / mL with antibody coating buffer, and add 100 μL of the diluted anti-CD28 antibody to each well of a 96-well black transparent bottom culture plate, and incubate at 4°C in the dark for 12 h. Discard the antibody liquid in the wells, add 200 μL of PBS containing 2% BSA to each well, and block at room temperature for 1 h; wash with PBS 3 times, each time stand for 5 min, discard the supernatant, and reserve for use.

[0140] 2. Preparation of CMTPX-labeled prostate cancer organoid suspension: centrifuge the prostate cancer organoids at 80 g for 3 min, discard the supernatant, and gently resuspend with PBS for 2 times. Dilute the CMTPX dye to a final concentration of 5 μM with prostate cancer culture medium, mix according to the ratio of 100 μL of dye solution per 1×10 4 prostate cancer organoids, incubate at 37°C in 5% CO2 for 30 min, add 1 mL of prostate cancer culture medium to terminate the incubation, centrifuge at 80 g for 3 min, discard the supernatant; resuspend the prostate cancer organoids with prostate cancer culture medium to a concentration of 2×10 3 cells / mL (calculated according to the effector-target ratio of 5:1, and 2×10 3Example 1-2 or Comparative Example 1-6 twice co-culture product 1 x 10 4 cells / mL;

[0141] 3, CFDASE-labeled twice co-culture product suspension preparation: centrifuge the twice co-culture product of Example 1-2 or Comparative Example 1-6 at 1500 rpm for 5 min, discard the supernatant, wash twice with PBS, resuspend to a concentration of 1 x 10 6 cells / mL; dilute the CFDASE dye to a final concentration of 5 μM with DMEM / F12 medium, mix with the CTL suspension at a volume ratio of 1:1, incubate at 37°C, 5% CO2for 20 min; add 5 times the volume of DMEM / F12 medium (containing 10% FBS), stand at room temperature for 10 min; centrifuge at 1500 rpm for 5 min, discard the supernatant; resuspend the CTL to a concentration of 1 x 10 4 cells / mL with DMEM / F12 medium.

[0142] 4, add 100 μL of CMTPX-labeled prostate cancer organoid suspension and 100 μL of CFDASE-labeled twice co-culture product suspension of Example 1-2 or Comparative Example 1-6 to each well of an anti-CD28-coated 96-well plate, and set up an organoid control group (100 μL of CMTPX-labeled prostate cancer organoid suspension + 100 μL of DMEM / F12 medium) as a control, incubate at 37°C, 5% CO2incubator for 72 h.

[0143] 5, observe the degree of red fluorescence quenching of prostate cancer organoids (killing effect) and the penetration of green fluorescence of twice co-culture product of Example 1-2 or Comparative Example 1-6 through the red area, analyze the imaging pictures at each time point with ImageJ software, select 3 random fields of view, and measure the red fluorescence integral optical density (IOD) of prostate cancer organoids and the green fluorescence IOD of twice co-culture product, respectively.

[0144] Organoid survival index = (killing group 72h red IOD / killing group 0h red IOD) x 100%;

[0145] Twice co-culture product proliferation index = (killing group 72h green IOD / killing group 0h green IOD) x 100%;

[0146] The lower the value of the organoid survival index, the stronger the killing effect; the higher the value of the twice co-culture product proliferation index, the more obvious the activation and proliferation.

[0147] The determination results are shown in Table 2:

[0148] Table 2

[0149]

[0150] Pharmacodynamic evaluation of ADC drug

[0151] The published prostate cancer ADC drug Sacituzumab govitecan (SG) was selected as the evaluation object to verify the effect of the organoid chip of embodiments 1-2 on the pharmacodynamic evaluation of ADC drugs. The two core pharmacodynamic indicators of organoid growth inhibition rate and cell proliferation inhibition rate were determined to verify the accuracy and reliability of the chip for ADC drug pharmacodynamic evaluation.

[0152] 1. Experimental method

[0153] Sacituzumab govitecan (SG) was gradiently diluted with sterile PBS buffer to prepare working solutions with concentrations of 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, and stored at 4°C in the dark for standby. The chip of embodiment 1 and the chip of embodiment 2 were both prepared according to the organoid chip of embodiments 1-2 and completed the preliminary co-culture process (to the second co-culture D12 stage).

[0154] The organoid chips of embodiments 1 and 2 were taken, and the PDO cell loading and drug loading were performed according to the use method of the organoid chip described in embodiment 1:

[0155] (1) PDO cell loading: The prostate cancer organoids in the base embodiment 1 were washed twice with sterile PBS buffer, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; the prostate cancer culture medium was used to adjust the concentration of the organoids to 2×10 5 cells / mL to obtain a PDO cell solution; 25 μL of PDO suspension (5×10 3 cells / well) was added to each co-culture well of the chip, and the chip was placed in a 37°C, 5% CO2 incubator for 2 h;

[0156] (2) Drug loading: 5 μL of SG drug solution with different concentrations was added to each co-culture well through the sample loading area, and a blank control group (sterile PBS buffer) was set up; after drug loading, the chip was placed back in a 37°C, 5% CO2 incubator for further culture for 48 h for pharmacodynamic evaluation.

[0157] 2. Experimental index

[0158] (1) Organoid growth inhibition rate detection

[0159] After the culture, each well of the organoids was dyed using an organoid live and dead staining kit: 100 μL of staining solution containing 2 μmol / L Calcein-AM and 4 μmol / L PI was added to each well, and incubation was performed at 37°C in the dark for 30 min; images were collected using a fluorescence microscope, and the survival area ratio of each well of the organoids was counted using ImageJ software to calculate the growth inhibition rate of the organoids under different drug concentrations.

[0160] Growth inhibition rate (%) = (1 - survival area ratio of the drug administration group of organoids / survival area ratio of the blank control group of organoids) x 100%.

[0161] (2) Cell proliferation inhibition rate detection

[0162] The above dyed chip was taken, the staining solution was discarded, 100 μL of prostate cancer culture medium containing 10% CCK-8 reagent was added to each well, and incubation was performed at 37°C and 5% CO2 for 2 h; the absorbance value (OD 450 ) at 450 nm wavelength was measured using a microplate reader, and the cell proliferation inhibition rate under different drug concentrations was calculated.

[0163] Proliferation inhibition rate (%) = (1 - OD 450 value of the drug administration group / OD 450 value of the blank control group) x 100%.

[0164] The determination results are shown in Table 3:

[0165] Table 3

[0166]

[0167] With the increase of the SG drug concentration, the growth inhibition rate and the cell proliferation inhibition rate of the prostate cancer organoids in the chips of Example 1 and Example 2 both showed a significant upward trend, and the inhibition rate was more than 80% when the concentration was 1 μmol / L, indicating that the organoid chip of the application can accurately reflect the concentration-effect relationship of the ADC drug, and meet the basic requirements of pharmacodynamic evaluation.

[0168] Compared with the traditional cell line model (without the participation of immune cells), the organoid chip provided by the application can simultaneously evaluate the direct killing of drugs on tumor cells and the regulatory effect on immune cells, providing an experimental platform closer to the real situation in vivo for the screening and efficacy prediction of ADC drugs, which helps to reduce the risk of clinical conversion of drugs.

[0169] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application shall be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. An organoid microarray for prostate cancer drug evaluation, characterized in that, The organoid chip includes a chip body, the chip body includes a co-culture region, the co-culture region is provided with co-culture wells, and the co-culture wells are filled with co-culture lyophilized reagents; The co-culture lyophilized reagent is obtained by lyophilizing a co-culture reagent. The co-culture reagent consists of IL-2, IL-15, anti-PD-L1 antibody, deferoxamine, HEPES, FGF-2, trehalose, mannitol, BSA, and culture medium, containing 15-20 IU / mL IL-2, 10-15 ng / mL IL-15, 3.5-5.0 μg / mL anti-PD-L1 antibody, 50-60 μmol / L deferoxamine, 10-15 mmol / L HEPES, 5-8 ng / mL FGF-2, 2%-3% w / v trehalose, 2%-5% w / v mannitol, and 0.1%-0.5% w / v BSA. The method of using the organoid chip includes: S1. Add organoid suspension and PBMC suspension to co-culture wells and perform initial co-culture under 10%-12% oxygen. S2. After the initial co-culture, discard the initial culture medium, add organoid suspension again, and carry out a second co-culture under 15%-18% oxygen to obtain the second co-culture product. S3. After co-culturing the secondary co-culture product with prostate cancer organoids, the ADC drug to be tested is added, and after culturing, it is used for drug efficacy evaluation.

2. The organ-on-a-chip according to claim 1, characterized in that, The co-culture reagent contains 20 IU / mL IL-2, 15 ng / mL IL-15, 5 μg / mL anti-PD-L1 antibody, 50 μmol / L deferoxamine, 10 mmol / L HEPES, 5 ng / mL LFGF-2, 2% w / v trehalose, 2% w / v mannitol and 0.1% w / v BSA.

3. The organ-on-a-chip according to claim 1, characterized in that, The organoid suspension mentioned in step S1 or step S2 is added according to the ratio of the number of organoids in the organoid suspension to the number of PBMC cells in the PBMC suspension; the ratio of the number of organoids in the organoid suspension to the number of PBMC cells in the PBMC suspension is 1:15-25. The prostate cancer organoids mentioned in step S3 are added according to the ratio of the number of prostate cancer organoids to the number of PBMC cells in the PBMC suspension; the ratio of the number of prostate cancer organoids to the number of PBMC cells in the PBMC suspension is 1:10-15.

4. The organ-on-a-chip according to claim 1, characterized in that, The initial co-culture conditions described in step S1 are 35-40℃, 5% CO2, 10%-12% oxygen, and culture for 7-9 days; The conditions for the secondary co-culture described in step S2 are 35-40℃, 5% CO2, 15%-18% oxygen, and culture for 5-7 days; The co-culture conditions described in step S3 are 35-40℃ and 5% CO2 for 2-5 hours; the culture conditions are 35-40℃ and 5% CO2 for 48-72 hours.

5. The organ-like chip according to claim 1, characterized in that, The organoid chip also includes a cell loading area, a sample loading area, and a waste liquid collection area, with each area connected by a microfluidic channel.

6. The method for preparing organoid chips according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: (1) Add the co-culture reagent to the co-culture wells, freeze-dry, and then cover the co-culture area with a sterile sealing film; (2) Assemble the cell loading area, co-culture area, sample loading area and waste collection area in sequence, with each area connected by a microfluidic channel; (3) After assembly, sterilize and connect oxygen sensor and temperature sensor to obtain organoid chip.

7. The application of the organ-on-a-chip according to any one of claims 1-5, characterized in that, The applications include any one or more of the following: (1) Application in the preparation of efficacy evaluation products for prostate cancer drugs; (2) Application in the efficacy evaluation of prostate cancer drugs.

8. A pharmacodynamic evaluation product for a prostate cancer drug, characterized in that, The efficacy evaluation product comprises the organoid chip as described in any one of claims 1-5.

9. A method for evaluating the efficacy of prostate cancer drugs, characterized in that, The method includes using organoid chips according to any one of claims 1-5 or the efficacy evaluation product according to claim 8.

Images