Construction method and application of pleuroperitoneal fluid-derived digestive tract tumor cell organ model

Abstract

The invention discloses a construction method of a primary cell organoid model and application of the primary cell organoid model in drug screening, the construction method comprises the following steps: (1) centrifuging malignant pleuroperitoneal fluid samples derived from digestive tract tumor patients, collecting cell precipitates, and preparing a single-cell suspension; (2) performing primary culture on the single-cell suspension to obtain primary culture cells; and (3) mixing the primary culture cells with matrigel, and carrying out organoid culture to obtain the primary cell organoid model. By adopting the method provided by the invention, the homogeneity of the organoids cultured by using the primary cells is higher, the number of successfully cultured organoids is more, and meanwhile, the related problems of limited passage and organoid amplification culture library building in the field of organoid culture are solved.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2023115152561 filed with the State Intellectual Property Office on November 15, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to a method for constructing a digestive tract tumor cell organoid model derived from pleural effusion and ascites, and an application thereof in drug screening. Background Art

[0004] Gastrointestinal cancer peritoneal metastasis (GICPM) is a major bottleneck problem in current clinical treatment. The cause of death in patients with advanced gastrointestinal tumors is often related to peritoneal metastasis cachexia. Peritoneal metastasis is a common metastatic site for gastrointestinal tumors including gastric cancer, colorectal cancer, biliary tumors, pancreatic cancer, etc. Patients with tumors with peritoneal metastasis often have a poor prognosis and a short overall survival (OS). Peritoneal metastasis is one of the common distant metastasis sites of gastric cancer. Studies have shown that peritoneal metastasis accounts for 50% of distant metastasis of gastric cancer. Patients with gastric cancer peritoneal metastasis have a very poor prognosis, with a median survival time of 7 to 10 months and a 5-year survival rate of less than 10%, with a median OS of only about 4 months, while the median OS of colorectal cancer peritoneal metastasis, which has a relatively good prognosis, is only about 12 months. At present, the recommended treatment mode for peritoneal metastasis of gastrointestinal tumors in both Eastern and Western treatment guidelines is systemic treatment, but the treatment effect is not ideal.

[0005] In recent years, with the promotion of clinical diagnosis and treatment technologies such as laparoscopic technology, intraperitoneal chemotherapy and hyperthermic intraperitoneal chemotherapy (HIPEC), the promotion of multidisciplinary treatment models, and the in-depth clinical and basic research in the field of peritoneal metastasis treatment, the overall prognosis of patients with peritoneal metastasis has improved to a certain extent compared with the past. Although some studies have tried peritoneal metastasis reduction surgery combined with hyperthermic intraperitoneal chemotherapy, which suggests that the overall survival may be prolonged, most of the studies are retrospective analyses, and preliminary conclusions show that patients undergoing peritoneal reduction surgery need to be strictly screened, and only a few patients can benefit from this method. Although the current second-generation high-throughput genome sequencing (NGS) can guide clinical drug selection to a certain extent, it has the characteristics of strong tumor heterogeneity and large individual differences among patients, which makes it impossible to quickly and effectively evaluate the efficacy of individualized drugs in order to accurately guide the selection of clinical drugs.

[0006] Organoids are tissue analogs with a certain spatial structure formed by three-dimensional (3D) culture of adult stem cells or pluripotent stem cells in vitro. Although organoids are not true human organs, they simulate real organs in structure and function, can simulate the structure and function of in vivo tissues to the greatest extent, and can be cultured for generations. At present, although the construction of patient-derived organoids (PDOs) based on patient tumors can be used for clinical drug screening to guide individualized precision treatment of patients, there are still some limitations in the construction and use of organoid models: (1) Limited passaging: In order to avoid the difference between the phenotype of tumor organoids after passaging and the in vivo situation, the passaging of tumor-derived organoids generally needs to be controlled within 2-3 generations, and no more than 5 generations at most; (2) Influence of mixed cells: Tumor tissues will also be mixed with some impurity cells such as fibroblasts, and their large-scale growth will affect the growth state of tumor organoids; (3) The construction of an organoid biobank requires a large sample size: The success rate of most organoid cultures is around 63-70%, and due to factors such as limited organoid passaging, a large sample size is required for library construction.

[0007] Zeng Yunli et al. have disclosed a method for culturing colorectal cancer organoids. However, this culture method obtains spherical cultures by directly culturing cell precipitates from the patient's ascites. The culture process does not go through the process of culturing primary seed cells, but directly cultures the separated cells into spherical cells (i.e., three-dimensional culture). A single separation can only prepare organoids once, and it is not possible to obtain "seed" primary cells that can sustainably culture organoids at the same time. Therefore, there are still defects in limited culture and the continuous need for a large sample size.

[0008] In addition, there is still no mature standard culture system for organoid culture based on malignant pleural effusions and ascites. The reason is that the tumor microenvironment of pleural effusions and ascites is significantly different from that of solid tumor tissues, the composition of body fluids in pleural effusions and ascites is very complex, and the responsiveness of different patients to drug treatment varies greatly. Most patients with advanced gastrointestinal tumors will develop uncontrollable malignant pleural effusions and ascites due to distant metastasis, which seriously affects the patient's survival prognosis. Therefore, the construction of a tumor organoid culture system derived from malignant pleural effusions of gastrointestinal tumors has extremely important application value for the research of gastrointestinal tumors and guiding personalized and precise treatment of patients. Summary of the invention

[0009] In order to solve at least some of the technical problems in the prior art, the present invention provides a method for constructing a primary cell organoid model and its application in drug screening. In order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0010] The first aspect of the present invention provides a method for constructing a primary cell organoid model, comprising the following steps:

[0011] (1) Centrifuge the pleural and ascites samples, collect the cell pellets, and prepare a single cell suspension;

[0012] (2) performing primary culture on the single cell suspension to obtain primary culture cells;

[0013] (3) The primary cultured cells are mixed into matrix gel for organoid culture to obtain a primary cell organoid model.

[0014] In a preferred embodiment, the culture medium for primary culture comprises the following components:

[0015] IMDM basal medium; 10% fetal bovine serum; 8-12 μM Y-27632; 0.05-0.15 w / w% penicillin-streptomycin; 0.05-0.15 w / w% β-mercaptoethanol and 0.5-1.5 w / w% MEM non-essential amino acid solution.

[0016] In another preferred embodiment, the culture medium for organoid culture comprises:

[0017] DMEM / F12 basal medium; 3%-5% fetal bovine serum; 50-100ng / mL EGF; 50-200ng / mL FGF-10; 50-200ng / mL R-Spondin-1; 50-150ng / mL Noggin; 50-100ng / mL Wnt-3α, 3-7w / w% KSR serum replacement; 8-12μM Y-27632; 0.05-0.15w / w% penicillin-streptomycin; 0.05-0.15w / w% β-mercaptoethanol; 5-1.5w / w% MEM non-essential amino acid solution.

[0018] Another aspect of the present invention also relates to the application of the organoid model constructed by the above construction method in drug screening.

[0019] In a preferred embodiment of the present invention, the drug refers to a chemotherapy drug, including but not limited to oxaliplatin, cisplatin, fluorouracil, capecitabine, paclitaxel, docetaxel and irinotecan.

[0020] The present invention has the following beneficial technical effects: :

[0021] 1. In the technical solution of the present invention, after the step of collecting the digestive tract tumor cell precipitation, a primary cell culture step is added. This step can achieve rapid expansion of tumor cells with stronger stemness from the mixed cells in the precipitation in a short period of time, which is conducive to the growth of tumor organoids and retains a large number of "seeds" for culturing organoids.

[0022] 2. The culture medium components for primary cell culture provided by the present invention help to improve the efficiency and cell viability of primary culture.

[0023] 3. The organoid culture medium components provided by the present invention help to improve the uniformity and quantity of organoids.

[0024] 4. The organoid model based on 3D culture technology of the present invention solves the defect that traditional 2D cultured primary cells lack tumor histological structural characteristics and therefore cannot fully reflect the tumor's responsiveness to drugs in vivo.

[0025] 5. The present invention solves the technical problem of the immaturity of the current organoid culture system based on pleural and ascites effusions.

[0026] 6. The culture system provided by the present invention can rapidly expand and purify primary cells and organoids derived from pleural and ascites in a short period of time. A large number of primary cells ensure sufficient "seeds" for the construction of organoids, which can effectively solve the defects of limited organoid passaging and changes in growth status after passaging. In addition, the organoid model constructed using primary cells has higher growth uniformity, more organoids, and better growth status, which is conducive to the construction of an organoid biological sample bank. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 a: Rendering of organoids cultured from primary cells derived from gastric cancer ascites samples;

[0028] Figure 1 b: Image of organoid cultured with cell pellets derived from gastric cancer ascites samples;

[0029] Figure 1 c: During the organoid culture stage, the number of organoids cultured directly using ascites precipitated cell samples was compared with the number of organoids cultured using ascites-derived primary cell samples;

[0030] Figure 2 :Primary cell culture, primary cell organoid culture and organoid pathology HE staining identification of ascites samples from different digestive tract tumors (gastric cancer, intestinal cancer, pancreatic cancer), showing that the organoids cultured in the primary cell organoid model construction system have typical cancer structures; F3P1 refers to the P1 generation organoids of the cultured F3 generation primary cells;

[0031] Figure 3 :A case study of the screening results of common chemotherapy drugs based on the primary cell organoid model derived from ascites of different gastric cancer patients (the relative percentage of live cells was tested after 96 hours of treatment with different drugs to quantify drug sensitivity). The figure shows that different patients have different sensitivities to different chemotherapy drugs. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

[0035] Example 1: Cultivation and construction of digestive tract tumor organoid model

[0036] 1.1 Preparation of reagents and consumables

[0037] (1) Consumables: 50 mL centrifuge tube, 70 μm sterile filter, 1.5 mL EP tube, 5 mL rubber-tipped dropper, 10 μL-1000 μL pipette tip, 24-well cell culture plate (Corning), 10 cm cell culture dish (Corning);

[0038] (2) Reagents: HBSS buffer (Gibco); DPBS balanced salt buffer (Gibco); 0.25% EDTA-trypsin (Gibco, Cat 25200072); Type II collagenase (Gibco, Cat: 17101-015); Matrigel (R&D systems, Cat: 3536-005-02); DMSO (Gibco) (3) Primary culture medium: IMDM basal medium (Iscove's Modified Dulbeccos's Medium, Gibco, Cat 12440053); 10% fetal bovine serum (Gibco,); 10 μM Y-27632 (PeproTech, Cat: 1293823); 0.1% penicillin-streptomycin (Gibco); 0.1% β-mercaptoethanol (Gibco, Cat: 21985023); 1% MEM non-essential amino acid solution (Gibco, Cat: 11140050);

[0039] (4) Organoid culture medium: DMEM / F12 basal medium (Gibco, Cat: 11330032); 3%-5% fetal bovine serum (Gibco); 50-100 ng / mL EGF (PeproTech, Cat: AF-100-15); 50-200 ng / mL FGF-10 (PeproTech, Cat: 100-26); 50-200 ng / mL Human R-Spondin-1 (PeproTech, Cat: 120-38); 50-150 ng / mL Noggin (PeproTech, Cat: 120-10C); 50-100 ng / mL Wnt-3α (PeproTech, Cat: 315-20); 5% Gibco TM Knock Out TM Serum replacement (KSR, Gibco, Cat: 10828028); 10 μM Y-27632 (PeproTech, Cat: 1293823); 0.1% penicillin-streptomycin (Gibco); 0.1% β-mercaptoethanol (Gibco, Cat: 21985023); 1% MEM non-essential amino acid solution (Gibco, Cat: 11140050);

[0040] (5) Cryopreservation solution: 10% DMSO + 90% fetal bovine serum.

[0041] 1.2 Pretreatment of pleural and ascites samples

[0042] (1) Treatment of pleural and ascites fluid: 100-300 mL of freshly isolated malignant pleural and ascites fluid samples from patients with digestive tract tumors (27 patients with malignant pleural and ascites fluid samples, all from Beijing Cancer Hospital, including 9 patients with gastric cancer, 9 patients with colorectal cancer, and 9 patients with pancreatic cancer. The sample collection was approved by the hospital ethics committee with the informed consent of the patients) were transferred into a 50 mL centrifuge tube under sterile conditions and incubated at 4°C, 2000 rpm for 5 min to collect the cell precipitate;

[0043] (2) Lyse red blood cells (choose whether to do this according to actual needs): Observe the cell pellet to see if there is obvious red blood cell precipitation. If so, treat the cells with 3-5 mL of RBC lysis buffer for 3-5 min, then use 5-10 mL of PBS to stop the red blood cell lysis, centrifuge at 2000 rpm for 5 min, and discard the supernatant. If red blood cell lysis is not required, proceed to the next step;

[0044] (3) Preparation of single cell suspension: (after lysis of red blood cells) add appropriate amount of DPBS buffer to resuspend the cell pellet, filter through a 70 μm filter to prepare a single cell suspension, then centrifuge at 2000 rpm for 5 min, discard the supernatant, and wash twice with DPBS buffer;

[0045] (4) Cell counting: Add an appropriate amount of DPBS buffer to resuspend the cells and use a cell counter to count the cells for later use.

[0046] 1.3 Primary cell culture

[0047] (1) Cell culture dish pretreatment

[0048] Dilute 50-100 μL of Matrigel in 5 mL of pre-cooled (0-4°C) DPBS, then transfer the 5 mL of the mixture to a 10 cm cell culture dish to evenly cover the entire bottom of the dish, and then place it in a 37°C cell culture incubator for more than 4-8 hours for later use;

[0049] (2) Primary cell culture

[0050] Take an appropriate amount (about 1x10 6 The primary cells prepared in 1.2 were inoculated into a 10 cm cell culture dish pre-coated with matrix gel, and then 8-10 mL of primary cell culture medium was added for primary cell culture and recorded as F0 generation, and cultured at 37°C, 5% CO2, and 95% relative saturated humidity;

[0051] (3) Primary cell culture medium replacement

[0052] The first inoculation culture (F0 generation) can be cultured for the first time 5-7 days after inoculation. After the culture is changed, continue to culture and observe the growth status;

[0053] (4) Primary cell passaging and cryopreservation

[0054] When the cell confluence reaches about 90%, the primary cells can be passaged. Collect the cultured primary cells using trypsin digestion and place them in a 15mL centrifuge tube, 1000rpm, 5min, discard the supernatant, and then passage them at a ratio of 1:2 (recorded as F1, 2, 3...n, n≤10, generally to ensure biological consistency with F0 generation samples, routine research uses cell samples within F5 generation) or freeze them at -80℃ gradient cooling and then transfer them to a liquid nitrogen tank for long-term storage.

[0055] 1.4 Organoid culture (direct ascites cell pellet culture and ascites primary cell culture)

[0056] (1) Organoid inoculation: According to the cell counting results, take an appropriate amount of pleural effusion cell pellet (F0) or primary cultured cells (F1-Fn), melt the cell pellet in Matrigel at 4°C overnight, and resuspend the cell pellet at 5-8×10 4 cells / point / well Inoculate the gel droplets mixed with cells into a 24-well cell culture plate (the matrix gel should be used on ice) and avoid bubbles during inoculation.

[0057] (2) Organoid culture: Place the 24-well plate inoculated with the cell-containing gel spheres upside down in a cell culture incubator. After the matrix gel is fully solidified (1-2 h), add 1 mL / well of organoid culture medium (see 1.1(4) above) and culture at 37°C, 5% CO2, and 95% relative saturated humidity.

[0058] (3) Organoid medium replacement: The culture status should be monitored in a timely manner during the sample culture period. The first medium change should be made after 5-7 days of culture. During the culture process, the growth status of the organoids should be observed and the medium should be changed every 3-5 days.

[0059] (4) Organoid passaging and cryopreservation: Discard the culture medium, add 200 μL of type II collagenase at a working concentration of 1-2 mg / mL to each well, and use a pipette to mechanically blow and place in an incubator at 37°C, 5% CO2 for digestion for about 30 minutes. Digest large pieces of organoids into small pieces suitable for expanded culture growth. After digestion, transfer them into 1.5 mL EP tubes with a pipette and then centrifuge at 2000 rpm for 5 minutes to obtain organoids suitable for culture. Wash them three times with organoid culture medium and seed them with matrix gel / cell spheres again (for specific operations, refer to step 1.4 (2)) or use organoid freezing solution to freeze at -80 degrees Celsius and then transfer them to a liquid nitrogen tank for long-term storage.

[0060] (5) Organoid histochemical identification: agar paraffin embedding, section HE staining (such as Figure 2(As shown) Pathological results showed that the cultured organoid samples all maintained the cancer tissue morphology of their tissue origin, proving that the organoid culture was successful. The cultured organoids can be used for clinical drug screening to guide personalized treatment for patients, or as a better in vitro research model for other preclinical studies.

[0061] Compared with direct culture of precipitated cells, organoid models constructed using primary cells cultured from patient ascites have better growth status; it was also observed that organoids cultured using primary cells have higher homogeneity and more successfully cultured organoids ( Figure 1 c).

[0062] Example 2 Drug Screening

[0063] 2.1 Sample sources and clinical data for constructing organoids:

[0064] Sample 1: Malignant pleural effusion in a patient with gastric cancer and pleural metastasis;

[0065] Sample 2: Malignant ascites in a patient with gastric cancer and peritoneal metastasis;

[0066] Sample 3: Malignant ascites in a patient with rectal cancer and peritoneal metastasis;

[0067] Sample 4: Malignant ascites in a patient with colon cancer and peritoneal metastasis.

[0068] (All patients were from Beijing Cancer Hospital, and sample collection was approved by the hospital ethics committee with the informed consent of the patients)

[0069] Samples 1-4 were all patients clinically diagnosed with advanced tumors (stage IV), and according to the patients' previous medical history data, patients 1 and 2 had received oxaliplatin treatment and had a certain control effect on the tumor; patients 3 and 4 had received albumin-paclitaxel intraperitoneal infusion chemotherapy, which also had a certain effect on ascites control.

[0070] The above samples 1-4 were cultured using the culture system described in Example 1 for model construction, and then subjected to drug screening tests respectively.

[0071] 2.2 Drug preparation

[0072]

[0073] 2.3 Screening steps

[0074] (1) The expanded and cultured organoid samples from pleural and ascites fluid were inoculated into a 96-well plate, with the number of organoids in each well controlled to be between 20 and 30, and 100 μL of culture medium in each well;

[0075] (2) Set up concentration gradients of 1000× and 10000×, with 3 biological replicates in each group, and add 100 μL of drug for treatment. At the same time, set up a blank control group without drug treatment, and the final culture system volume of each well was 200 uL;

[0076] (3) Routine culture: Organoid proliferation activity detection is performed on the 4th to 6th day after drug treatment. Drug sensitivity is evaluated based on organoid vitality. The specific steps of organoid proliferation activity detection are as follows:

[0077] ①Detection reagents:

[0078] Choose CellTiter-Lumi TM Plus luminescent cell viability detection kit (Biyuntian C0068, CellTiter-Lumi TM Plus Luminescent Cell Viability Detection Kit) is used to detect the proliferation of cells in the sample.

[0079] ②Cell viability detection

[0080] a. Take out the cell culture plate and equilibrate it at room temperature for 10-15 minutes.

[0081] b. Add 50-100 μL CellTiter-Lumi TM Plus luminescence detection reagent.

[0082] c. Shake at room temperature for 2 minutes to promote cell lysis.

[0083] d. Incubate at room temperature (about 25°C) for 10-15 minutes to allow the luminescent signal to stabilize.

[0084] e. Then transfer to a 96-well plate suitable for chemiluminescence detection, and set up a culture medium well without cells as a negative control.

[0085] f. Use a multifunctional microplate reader with chemiluminescence detection function to perform chemiluminescence detection.

[0086] g. Calculate the relative viability of cells directly based on chemiluminescence readings.

[0087] 2.4 Screening results:

[0088] The results of drug susceptibility assessment were as follows Figure 3 The results showed that in case 1, the samples from malignant pleural effusion of patients with gastric cancer and pleural metastasis were mainly sensitive to 5-fluorouracil; in case 2, the samples from malignant ascites of patients with gastric cancer and peritoneal metastasis were mainly sensitive to cisplatin; in case 3, the samples from malignant ascites of patients with rectal cancer and peritoneal metastasis were mainly sensitive to irinotecan; in case 4, the samples from malignant ascites of patients with colon cancer and peritoneal metastasis were mainly sensitive to paclitaxel.

[0089] By comparing the patient's previous medical history data, it can be found that the organoid model constructed according to the culture system provided by the present invention may be used for clinical drug screening to guide the feasibility of individualized treatment.

[0090] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A method for constructing a primary cell organoid model, characterized in that: The steps include: (1) The malignant pleural and ascites samples from patients with digestive tract tumors were centrifuged, the cell pellets were collected, and single cell suspensions were prepared; (2) Primary culture of the single cell suspension to obtain primary culture cells derived from malignant pleural effusion and ascites of digestive tract tumors; (3) mixing primary cultured cells into matrix gel for organoid culture to obtain a primary cell organoid model; The culture medium for primary culture comprises: IMDM basic culture medium; 10% fetal bovine serum; 8-12 μM Y-27632; 0.05-0.15 w / w% penicillin-streptomycin; 0.05-0.15 w / w% β-mercaptoethanol and 0.5-1.5 w / w% MEM non-essential amino acid solution; The primary culture method comprises: (a) Cell culture dish pretreatment Dilute the matrix gel in pre-cooled DPBS, then transfer the DPBS mixture to the cell culture dish to evenly cover the entire bottom of the dish, and then place it in a 37°C cell culture incubator for more than 4-8 hours for later use; (b) Primary cell culture Taking an appropriate amount of the primary cells prepared in step (a) and inoculating them in a cell culture dish pretreated and coated with matrix gel, then adding the primary culture medium to culture the primary cells and recording them as F0 generation, and culturing them at 37° C., 5% CO2, and 95% relative saturated humidity; (c) Primary cell culture medium replacement For the first inoculation culture, the first medium change can be made 5-7 days after inoculation. After the medium change, continue to culture and observe the growth status; (d) Primary cell passaging and cryopreservation When the cell confluence reaches about 90%, the primary cells can be subcultured. The cultured primary cells are collected by trypsin digestion and placed in a centrifuge tube at 1000 rpm for 5 min. The supernatant is discarded and then subcultured at a ratio of 1:2 or cryopreserved at -80°C and then transferred to a liquid nitrogen tank for long-term storage. The culture medium for organoid culture includes: DMEM / F12 basal medium; 3%-5% fetal bovine serum; 50-100ng / mL EGF; 50-200ng / mL FGF-10; 50-200ng / mL R-Spondin-1; 50-150ng / mL Noggin; 50-100ng / mL Wnt-3α, 3-7w / w% KSR serum replacement; 8-12μMY-27632; 0.05-0.15w / w% penicillin-streptomycin; 0.05-0.15w / w% β-mercaptoethanol; 5-1.5w / w% MEM non-essential amino acid solution.

2. The construction method according to claim 1, characterized in that: After collecting the cell pellet, a step of lysing the red blood cells is also included.

3. Application of the method for constructing the organoid model described in claims 1-2 in drug screening.

4. The use according to claim 3, wherein the drug is a chemotherapy drug.

5. The use according to claim 4, wherein the chemotherapy drugs include oxaliplatin, cisplatin, fluorouracil, capecitabine, paclitaxel, docetaxel and irinotecan.

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