Organ-like culture method and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses a culture method and application of an organoid. The culture method is easy to operate and high in success rate. The organoid constructed by the method can be used for constructing an in-vitro organoid and peripheral blood lymphocyte co-culture system to evaluate the curative effect of tumor immunotherapy, the co-culture system predicts that AUC which is possibly invalid in immunotherapy is 0.82 (95% CI, 0.71-0.93), the sensitivity can reach 90.90%, the specificity is 73.50%, the positive predictive value is 77.42%, and the negative predictive value is 88.98%, which are obviously higher than PD-L1 (the sensitivity is 63.60%; the specificity is 37.20%), a new screening index is provided for determination of a precise immunotherapy scheme, and the treatment effect of a patient is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for culturing an organoid and an application thereof. Background Art

[0002] Lung cancer, a malignant disease characterized by significant individual variability and tumor heterogeneity, exhibits high heterogeneity in clinical presentation, histological features, and epigenetic profiles. This biological characteristic has directly driven a shift in clinical treatment towards personalized precision medicine. Patient-derived organoids (PDOs), due to their ability to highly retain the characteristics of the patient's tumor microenvironment, have become a crucial in vitro research model for precision medicine. However, current lung cancer PDO culture systems face technical bottlenecks, with an overall success rate ranging from 32% to 78%, with adenocarcinomas having a higher success rate than squamous cell carcinomas. Furthermore, depending on tissue source, the success rate for surgical specimens (58±12%) is higher than for needle biopsies (34±9%) and higher than for pleural effusions (27±15%), severely limiting the translational application of PDO models. Literature has reported that optimizing the culture medium can significantly improve the success rate of organoid culture.

[0003] Conditional reprogramming (CR) technology allows for rapid and stable expansion of tumor cells in vitro, with a high degree of retention of the genotype and phenotype of tumor cells. This allows researchers to screen for sensitivity to tumor immunotherapy quickly enough, thereby providing information for clinical use. However, traditional conditional reprogramming technology has some shortcomings, such as the need to add 3T3-J2 feeder cells during the culture process, which makes passaging and other processes very cumbersome. Therefore, it is very necessary to improve conditional reprogramming technology and apply it to the culture of organoids.

[0004] Immunotherapy, as an important cancer treatment, has shown tremendous potential in clinical application. However, accurate and effective methods are currently lacking for assessing the efficacy of individual immunotherapy patients. Overall, the efficacy of anti-PD-1 / PD-L1 therapy in cancer patients is only approximately 20%. Therefore, identifying effective biomarkers to accurately identify patients who will benefit has become a hot topic in immunotherapy research in recent years.

[0005] Organoid culture alone cannot simulate the in vivo immune microenvironment, making it difficult to accurately assess the efficacy of immunotherapy. Therefore, an innovative approach is needed to address this issue. Co-culturing organoids with immune cells enhances the environmental stimulation of immune cell infiltration during in vitro organoid culture, more closely resembling the in vivo growth environment. This provides an ideal experimental platform for studying autoimmune diseases, tumors, and related immunotherapies. Summary of the Invention

[0006] The purpose of the present invention is to overcome at least one deficiency of the prior art, provide a method for culturing organoids and its application, and establish a co-culture platform of organoids and peripheral blood mononuclear cells for evaluating the efficacy of immunotherapy.

[0007] The technical solution adopted by the present invention is: A method for culturing lung tumor organoids, comprising: Prepare conditioned medium: The conditioned medium is a basal medium supplemented with 4.5-5.5 v / v% fetal bovine serum, 23-28 μg / mL hydrocortisone, 0.120-0.130 μg / mL EGF, 4.5-5.5 μg / mL insulin, 9-11 nM Y-27632, and appropriate amounts of antibiotics; Collecting the supernatant: 3T3-J2 fibroblasts were irradiated with a non-lethal dose of X-rays, and then cultured in the conditioned medium. After culturing for 48 to 72 hours, the supernatant was collected. Prepare improved culture medium: add 4.5-5.5 μg / mL A83-01 and 4.5-5.5 μg / mL CHIR9902 to the supernatant to obtain improved culture medium; Organoid model establishment: Lung cancer cells obtained by digestion and separation of lung cancer tissue are added to the modified culture medium and resuspended. Matrigel is added and gently mixed. The cells are then injected into a well plate and incubated. After the Matrigel solidifies, the modified culture medium is added. The modified culture medium is replaced every 3-4 days to obtain an organoid model.

[0008] In some examples, the basal culture medium is a mixed culture medium of DMEM and F12 culture medium.

[0009] In some examples, the basal culture medium is DMEM and F12 culture medium in a volume mixing ratio of (2.5-3.5):1.

[0010] In some examples, the antibiotics consist of 1% penicillin / streptomycin, 10 μg / mL gentamicin, 250 ng / mL amphotericin B, and 8.6 ng / mL cholera toxin.

[0011] In some embodiments, the X-ray dose is 36-44 Gray.

[0012] In some examples, when establishing an organoid model, the volume mixing ratio of the modified culture medium to the matrix gel is 1: (2.5-3.5).

[0013] In some examples, organoid models are passaged as follows: Aspirate the old modified culture medium, then slowly add PBS along the wall of the well to wash twice, then add TrypLE enzyme to blow away the matrix gel, digest at 37℃ for 10 minutes to turn it into single cells again, and resuspend and passage at a ratio of 1 to 4 after centrifugation.

[0014] In some examples, organoid models are cryopreserved as follows: Aspirate the old modified culture medium, then slowly add PBS along the wall of the well to wash twice, then add PBS pre-chilled at 4℃ to disperse the matrix gel, incubate at 4℃ for 30min to allow the matrix gel to fully dissolve in PBS, then centrifuge at 4℃ 1000 r / min for 5min, discard the supernatant and add freezing solution, then cool through program, and finally transfer to liquid nitrogen for ultra-low temperature storage.

[0015] The above features can be combined arbitrarily unless they conflict with each other.

[0016] The second aspect of the present invention provides: The first aspect of the present invention relates to the use of lung tumor organoids in evaluating the efficacy of immunotherapy in patients.

[0017] In some examples, the immunotherapy is an immune checkpoint inhibitor - anti-PD-1 / PD-L1 therapy.

[0018] In some embodiments, the evaluation includes the following steps: Extraction of mononuclear cells from the patient's peripheral blood: Mononuclear cells are extracted from the peripheral blood of patients with tumor tissue and set aside; Co-culture preparation: Stimulate organoids with 200 ng / mL IFN-γ overnight, then coat the plates with 100 μL of 5 μg / mL CD3 antibody at 4°C overnight. Co-culture of organoids and lymphocytes: Remove the organoid culture medium, rinse with PBS, digest with TrypLE, terminate the digestion with conditioned medium, centrifuge, count, and set aside. Mix the effector peripheral blood mononuclear cells and organoid cells at a ratio of 20:1. Add the suspension to a CD3 antibody-coated culture plate and add 20 μg / mL anti-PD-1 antibody to co-culture at 37°C, 5% CO2 for 24 hours. Post-coculture evaluation: The co-culture supernatant was aspirated and T cell function-related factors IFN-γ, TNF-α, and IL-6 were detected by flow cytometry; live and dead cell levels were analyzed using double staining. The efficacy of immunotherapy in patients was determined based on the co-culture situation.

[0019] The third aspect of the present invention provides: A co-culture device for co-culturing the lung tumor organoids described in the first aspect of the present invention, mononuclear cells extracted from the patient's peripheral blood, and an immunotherapy preparation; A detection device for determining the expression levels of T cell function-related factors IFN-γ, TNF-α and IL-6 and the levels of live and dead cells after co-culture; The result analysis device determines the efficacy of the patient's immunotherapy based on the detection results of the detection device.

[0020] In some examples, the detection device is a flow analyzer.

[0021] The beneficial effects of the present invention are: In some examples of the present invention, an in vitro organoid and peripheral blood lymphocyte co-culture system can be constructed to evaluate the efficacy of tumor immunotherapy. The AUC of this co-culture system for predicting the possible ineffectiveness of immunotherapy is 0.82 (95% CI, 0.71-0.93), with a sensitivity of 90.90%, a specificity of 73.50%, a positive predictive value of 77.42%, and a negative predictive value of 88.98%, which are significantly higher than PD-L1 (sensitivity: 63.60%; specificity: 37.20%).

[0022] Some examples of the tumor immunotherapy efficacy evaluation system of the present invention can be used to pre-evaluate the efficacy of immunotherapy for tumor patients before treatment, provide new screening indicators for determining precise immunotherapy plans, and improve the treatment effect of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The morphology of non-small cell lung cancer organoids cultured under improved reprogramming conditions was counted: including HE staining of organoid-derived tissue, HE staining of organoids, and bright field images of organoids.

[0024] Figure 2 Immunofluorescence analysis of non-small cell lung cancer organoids cultured using modified conditional reprogramming technology. TTF-1 is a marker for lung adenocarcinoma, and P63 is a marker for lung squamous cell carcinoma.

[0025] Figure 3 This is a comparison of tumor susceptibility genes in three samples of different passage organoids (PDOs) and source tumor tissues from patients with non-small cell lung cancer (NSCLCs).

[0026] Figure 4 The number of mutations in each gene in three samples of organoids (PDOs) of different passages and the source tumor tissues from patients with non-small cell lung cancer (NSCLCs).

[0027] Figure 5 This study evaluated the efficacy of a co-culture system of organoids and peripheral blood mononuclear cells. Dual staining of live and dead cells revealed a significant increase in dead cells when PD-1 antibodies were added to organoids from patients who responded to treatment.

[0028] Figure 6 This study evaluated the efficacy of a co-culture system of organoids and peripheral blood mononuclear cells. A. T cells from patients who responded to treatment significantly increased secretion of IFN-γ, TNF-α, and IL-6. B. T cells from patients who did not respond to treatment significantly increased secretion of IFN-γ, TNF-α, and IL-6.

[0029] Figure 7 Figure 3 shows the receiver operating characteristic (ROC) of the organoid and peripheral blood mononuclear cell co-culture system. As can be seen, the AUC for this system in predicting the likelihood of immunotherapy failure is 0.82 (95% CI, 0.71-0.93).

[0030] Figure 8 The co-culture system of organoids and peripheral blood mononuclear cells is associated with patient prognosis. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further illustrated below in conjunction with experiments.

[0032] 1. Tissue specimens from lung cancer surgical resections at our hospital were collected, and pathological diagnosis of lung adenocarcinoma was confirmed. Clinical data (name, sex, age, diagnosis, time of initiation of subsequent PD-1 therapy, PD-1 therapy response, TNM stage, and PD-L1 expression (TPS)) were collected.

[0033] Table 1. Demographics and baseline clinical characteristics of patients 2. Preparation of conditioned medium: DMEM / F12 (v / v, 3:1) medium supplemented with 5% fetal bovine serum, 1% penicillin / streptomycin, 25 μg / mL hydrocortisone, 0.125 μg / mL EGF, 10 μg / mL gentamicin, 250 ng / mL amphotericin B, 5 μg / mL insulin, 8.6 ng / mL cholera toxin, and 10 nM Y-27632.

[0034] 3. Preparation of feeder cells: 3T3-J2 fibroblasts were irradiated with 40 Gray X-rays.

[0035] 4. Preparation of modified culture medium: 5×10 6 Feeder layer cells were plated in a 10 cm culture dish, conditioned medium was added and the dish was cultured in a 37°C, 5% CO2 incubator for 48 hours. The supernatant was collected and then A83-01 and CHIR9902 were added.

[0036] 5. Isolation of primary non-small cell lung cancer cells: Place the non-small cell lung cancer tissue removed from surgery in DMEM containing 5% fetal bovine serum and 1% double-antibody, place it in an ice box and transport it back to the laboratory. After removing necrotic tissue and blood clots, rinse it with PBS several times, and then cut it into 1 mm pieces in a 5 mL eppendorf tube. 2 The fragments were separated and an appropriate amount of 0.1% type IV collagenase was added. The cells were digested in a 37°C water bath for 1 h, and then the culture medium was added to terminate the digestion. The cells were filtered through a 100 μm filter, and the filtrate was centrifuged at 1000 r / min for 5 min. The supernatant was discarded and set aside.

[0037] 6. Organoid Model Establishment: Add 50 μL of conditioned medium to the above cell pellet and resuspend it. Then add 150 μL of Matrigel and gently mix. Pipette 20 μL at a time into a 6-well plate and incubate in an incubator for 20 min. After the Matrigel solidifies, add 4 mL of conditioned medium. Change the culture medium every 3-4 days. Organoid formation can be seen in about 4 days, and it can be passaged after 10 days.

[0038] 7. Identification of organoid models: light microscopy and HE staining of organoids ( Figure 1 ), identification of organoids by immunofluorescence ( Figure 2 To further verify that organoids can retain the gene mutations of parental tissues, we performed whole-exome sequencing on three lung adenocarcinoma organoids and their paired parental tissues. The results confirmed that the gene mutation sites, mutation types, and number of chromosomal mutations in organoids and their derived tumor tissues were highly consistent ( Figure 3 , Figure 4 ).

[0039] 8. Passaging the organoid model: Aspirate the old culture medium and slowly add PBS along the wall of the well to wash twice. Then add TrypLE enzyme to blow away the matrix gel. Digest at 37°C for 10 minutes to convert the cells back into single cells. After centrifugation, resuspend and passage at a ratio of 1 to 4.

[0040] 9. Cryopreservation of organoid models: Aspirate the old culture medium and slowly add PBS along the wall of the well to wash twice. Then, add PBS that has been pre-chilled at 4°C to disperse the Matrigel. Allow the Matrigel to fully dissolve in PBS at 4°C for 30 minutes. Centrifuge at 1000 rpm at 4°C for 5 minutes. Discard the supernatant and add freezing solution. Then cool the well through a program and finally transfer to liquid nitrogen for ultra-low temperature storage.

[0041] Efficacy prediction: 1. Extraction of peripheral blood mononuclear cells from patients: 3 mL of peripheral blood was extracted from patients with tumor tissue, and the peripheral blood mononuclear cells were separated using Ficoll separation solution and frozen at -80°C for later use.

[0042] 2. Co-culture Preparation: Stimulate organoids with 200 ng / mL IFN-γ overnight, thaw frozen peripheral blood mononuclear cells overnight, and coat the wells of a 96-well U-shaped plate with 100 μL of 5 μg / mL CD3 antibody at 4°C overnight.

[0043] 3. Co-culture of Organoids with Lymphocytes: On the second day, organoids were processed by removing culture medium, rinsing with PBS, digesting with TrypLE, terminating digestion with conditioned medium, centrifuging, and counting. Resuscitated peripheral blood mononuclear cells were also counted. After mixing effector peripheral blood mononuclear cells with organoids at a ratio of 20:1, the suspension was transferred to a 96-well U-shaped plate. Anti-PD-1 antibody (20 μg / mL) was added and incubated at 37°C, 5% CO₂ for 48 hours.

[0044] 4. Evaluation after co-culture: Calcein-AM and PI dual dyes were used to perform dual staining of live and dead cells. The levels of live and dead cells were analyzed. It can be seen that when PD-1 antibodies were added to the organoids of patients with effective treatment, the number of dead cells increased significantly ( Figure 5 , samples 1 and 2), no significant changes were observed in organoids from patients with ineffective treatment ( Figure 5 , sample 3); the co-culture supernatant was aspirated and T cell function-related factors IFN-γ, TNF-α and IL-6 were detected by flow cytometry. It can be seen that the secretion of IFN-γ, TNF-α and IL-6 by T cells of patients with effective treatment was significantly increased ( Figure 6 A) T cells from patients with unresponsiveness to treatment showed no significant changes in IFN-γ, TNF-α, and IL-6 secretion ( Figure 6 B).

[0045] 5. Comparison of the predictive effects of different assessments in combination with clinical efficacy: Comparison of the prediction results of the co-culture assessment system with the actual clinical efficacy results (Table 2). The organoid and peripheral blood mononuclear cell co-culture system has a sensitivity of 90.90%, a specificity of 73.50%, a positive predictive value of 77.42%, and a negative predictive value of 88.98%. This is significantly higher than PD-L1 (sensitivity: 63.60%; specificity: 37.20%), as shown in Table 3. The AUC of this system for predicting the likelihood of immunotherapy failure is 0.82 (95% CI, 0.71-0.93). Figure 7 ).

[0046] Table 2. Comparison of the co-culture evaluation system and actual clinical efficacy prediction results Table 3. Comparison of different prediction methods 6. Kaplan-Meier analysis found that organoid co-culture system evaluation was closely related to patient OS, and patients with ineffective evaluation had a poor prognosis ( Figure 8 ), suggesting that the organoid co-culture system can be used to predict the efficacy of ICI treatment.

[0047] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A method for culturing lung tumor organoids, characterized in that: include: Prepare conditioned medium: The conditioned medium is a basal medium supplemented with 4.5-5.5 v / v% fetal bovine serum, 23-28 μg / mL hydrocortisone, 0.120-0.130 μg / mL EGF, 4.5-5.5 μg / mL insulin, 9-11 nM Y-27632, and appropriate amounts of antibiotics; Collecting the supernatant: 3T3-J2 fibroblasts were irradiated with a non-lethal dose of X-rays, and then cultured in the conditioned medium. After culturing for 48 to 72 hours, the supernatant was collected. Prepare improved culture medium: add 4.5-5.5 μg / mL A83-01 and 4.5-5.5 μg / mL CHIR9902 to the supernatant to obtain improved culture medium; Organoid model establishment: Lung cancer cells obtained by digestion and separation of lung cancer tissue are added to the modified culture medium and resuspended. Matrigel is added and gently mixed. The cells are then injected into a well plate and incubated. After the Matrigel solidifies, the modified culture medium is added. The modified culture medium is replaced every 3-4 days to obtain an organoid model.

2. The culture method according to claim 1, wherein The basic culture medium is a mixed culture medium of DMEM and F12 culture medium.

3. The culture method according to claim 2, wherein The basic culture medium is DMEM and F12 culture medium with a volume mixing ratio of (2.5-3.5):

1.

4. The culture method according to any one of claims 1 to 3, characterized in that The antibiotics consisted of 1% penicillin / streptomycin, 10 μg / mL gentamicin, 250 ng / mL amphotericin B, and 8.6 ng / mL cholera toxin.

5. The culture method according to any one of claims 1 to 3, characterized in that The X-ray radiation dose is 36 to 44 Gray.

6. The culture method according to any one of claims 1 to 3, characterized in that In the establishment of organoid models, the volume mixing ratio of modified culture medium and matrix gel is 1: (2.5~3.5).

7. Use of the lung tumor organoid according to any one of claims 1 to 6 in evaluating the efficacy of immunotherapy in patients.

8. The use according to claim 7, characterized in that The immunotherapy is an immune checkpoint inhibitor-anti-PD-1 / PD-L1 treatment.

9. The use according to claim 7, characterized in that The assessment includes the following steps: Extraction of mononuclear cells from the patient's peripheral blood: Mononuclear cells are extracted from the peripheral blood of patients with tumor tissue and set aside; Co-culture preparation: Organoids were stimulated with 200 ng / mL IFN-γ overnight, and then coated with 100 μL of 5 μg / mL LCD3 antibody at 4°C overnight. Co-culture of organoids and lymphocytes: Remove the organoid culture medium, rinse with PBS, digest with TrypLE, terminate the digestion with conditioned medium, centrifuge, count, and set aside. Mix the effector peripheral blood mononuclear cells and organoid cells at a ratio of 20:

1. Add the suspension to a culture plate coated with CD3 antibody and add 20 μg / mL anti-PD-1 antibody. Co-culture at 37°C, 5% CO2 for 24 hours. Post-coculture evaluation: The co-culture supernatant was aspirated and T cell function-related factors IFN-γ, TNF-α, and IL-6 were detected by flow cytometry; live and dead cell levels were analyzed using double staining. The efficacy of immunotherapy in patients was determined based on the co-culture situation.

10. A system for evaluating the efficacy of immunotherapy in a patient, comprising: A co-culture device for co-culturing the lung tumor organoid according to any one of claims 1 to 6, mononuclear cells extracted from the patient's peripheral blood, and an immunotherapy preparation; A detection device for determining the expression levels of T cell function-related factors IFN-γ, TNF-α and IL-6 and the levels of live and dead cells after co-culture; The result analysis device determines the efficacy of the patient's immunotherapy based on the detection results of the detection device.

Images