Tumor organ-immune cell co-culture method and application
Through co-culture of tumor organoids and T cells and flow cytometry detection, the problem of difficult to accurately evaluate drug sensitivity and dependence on experimental animals in the prior art is solved, personalized treatment and efficient screening are achieved, and experimental animals are reduced. dependency.
Patent Information
- Application Number
- CN202411454811.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2025-02-14
AI Technical Summary
Existing pharmacodynamic testing methods for individual tumors are difficult to accurately evaluate the sensitivity of drugs to individual patients, and rely on experimental animals, which have ethical and environmental problems.
By co-culturing tumor organoids and T cells, flow cytometry is used to detect the sensitivity of different drug use regimens, and the killing of different drugs to organoids is detected, thereby reflecting the sensitivity of the patient's autologous tissue to the drug use regimen.
It realizes personalized assessment of drug sensitivity, reduces dependence on experimental animals, meets ethical and environmental protection requirements, and has high-throughput, high-resolution screening capabilities.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedicine, and more specifically, to a method and application of tumor organoid-immune cell co-culture. Background Art
[0002] Organoids are micro-organs with three-dimensional structures that are cultured in vitro based on the simulation of real human organs and physiological systems. They are accelerated in the field of clinical drug research because they have complex structures similar to real organs and can partially simulate the physiological functions of the source tissues and organs. Specifically, they are based on fresh tumor tissue removed or biopsied from patients, and are cultured in an in vitro 3D environment to form an in vitro model that is highly similar to the patient's tumor characteristics. Organoids have the ability to self-renew and maintain the physiological structure and function of the source tissue. They are known as "micro-organs in a culture dish" and are highly complex. They are currently the research model that is closest to the in vivo state.
[0003] Organoids are the latest and internationally leading method for testing the pharmacodynamics of individual tumors. They use organoids cultured from the patient's own tissue to conduct sensitivity tests on possible therapeutic drugs or regimens, and can very accurately predict the effectiveness of clinical treatment. Summary of the invention
[0004] The purpose of the invention of this application is to co-culture organoids with T cells, use flow cytometry to detect the sensitivity of different medication regimens, and detect the lethality of different drugs to organoids, thereby reflecting the sensitivity of the patient's autologous tissue to the medication regimen.
[0005] In order to achieve the above invention objectives, this application adopts the following technical solutions:
[0006] In a first aspect, the present application discloses a method for co-culturing tumor organoids and immune cells, comprising the following steps:
[0007] S1, Organoid digestion and counting;
[0008] S2, T cell expansion;
[0009] S3, T cell count;
[0010] S4, calculation of cell suspension volume;
[0011] S5, coculture;
[0012] S51, according to the volume calculated in step S4, the organoid cell suspension and the T cell suspension are mixed, the number of organoid cells is 1000-1500 per well, and the number of T cells is 10000-15000 per well;
[0013] S52, centrifugation, 1500 rpm, 5 min;
[0014] S53, discard the supernatant after centrifugation, add the corresponding Matrigel and co-culture medium according to the experimental protocol, and Matrigel is 10 μL per well;
[0015] S54, Matrigel and 90 μL of cells were mixed and evenly seeded in 96-well plates for co-culture.
[0016] Furthermore, step S1 includes the following steps:
[0017] Observe the state of the 48-well plate organoids under a microscope. When there are 2,000 to 3,000 organoids in each protocol, co-culture with immune cells.
[0018] Discard the supernatant from the wells in the well plate where the experiment is to be conducted, add 200 μL of Tryple for digestion, and gently pipette with a 200 μL pipette until most of the organoids are in the form of microspheres;
[0019] After digestion was complete, 200 μL of washing solution was added to terminate the digestion;
[0020] Use a 200μL pipette to transfer the cell suspension in the 48-well plate to a 1.5ml EP tube;
[0021] Take 10 μL of single cell suspension and 10 μL of AOPI dye solution and mix them respectively, and then take 20 μL of the mixture to count with a cell counter.
[0022] Further, step S2 includes the following steps:
[0023] In a 15 mL centrifuge tube, add Hank's Buffer in an equal volume to the blood, then add the blood sample and mix gently to obtain a mixed solution;
[0024] Add lymphocyte separation solution to a 50 mL centrifuge tube, and add the mixed solution in the above step dropwise onto the upper layer of lymphocyte separation solution along the wall one second at a time. The volume ratio of lymphocyte separation solution to blood sample is 2:1.
[0025] Centrifuge at 4°C, 2000 rpm, 20 min;
[0026] The PBMC layer was aspirated, washed with 1 mL PBS, centrifuged at 1500 rpm for 5 min at 4°C, repeated washing 3 times, and counted;
[0027] T cells were cultured in suspension using T cell culture medium and expanded. When the number of cells expanded to 10 times that of organoids, they were co-cultured with organoids in a carbon dioxide incubator at 37°C for 5 days.
[0028] Furthermore, the T cell seeding density is 2000 to 10000 per well.
[0029] Further, step S3 includes the following steps:
[0030] Observe the status of T cells in the 48-well plate under a microscope to determine whether co-culture is possible;
[0031] Transfer the cell suspension in the wells to be tested in the 48-well plate to a 1.5 ml EP tube;
[0032] 10 μL of T cell suspension and AOPI were mixed respectively, and 20 μL of the mixture was counted using a cell counter.
[0033] Further, step S3 includes the following steps:
[0034] Calculate the concentration of organoid cell suspension and T cell suspension based on the cell counting results;
[0035] Calculate the required volumes of organoid cell suspension and T cell suspension so that the ratio of T cells to organoid cells in each well is 10:1.
[0036] Further, in step S53, the co-culture medium is half organoid medium and half T cell medium.
[0037] In a second aspect, the present application provides the use of the method described in the first aspect in preparing a product for detecting a patient's sensitivity to a medication.
[0038] Furthermore, the product includes a detection kit.
[0039] The present application discovered that by co-culturing organoids with T cells, flow cytometry was used to detect the sensitivity of different medication regimens and the lethality of different drugs to organoids, thereby reflecting the sensitivity of the patient's autologous tissue to the medication regimen.
[0040] In summary, compared with the prior art, this application has the following advantages:
[0041] (1) Personalized treatment: By simulating the patient's own tissue, the sensitivity of the drug to the individual patient can be more accurately evaluated, providing a basis for personalized treatment.
[0042] (2) Efficient screening: Flow cytometry has the characteristics of high throughput and high resolution, which can quickly screen out effective drug combinations and medication regimens.
[0043] (3) Reduce the use of experimental animals: Drug screening through organoid models can reduce dependence on experimental animals and comply with ethical and environmental requirements.
[0044] (4) Broad application prospects: The method of the present application has good application prospects in the fields of tumor treatment, immune system disease treatment, drug development, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 : The drug sensitivity test results of Experiment 1 in Example 2 ( Figure 1 A is the result graph of the control (DMSO); Figure 1 B is the result diagram of scheme 1; Figure 1 C is the result graph of Scheme 2; Figure 1 D is the result diagram of scheme 3; Figure 1 E is the result graph of Scheme 4; Figure 1 F is the result graph of Scheme 5; Figure 1 G is the result graph of Scheme 6; Figure 1 H is the result graph of Scheme 7; Figure 1 I is the result diagram of Scheme 8);
[0046] Figure 2 : The drug sensitivity test results of Experiment 2 in Example 2 ( Figure 2 A is the result graph of the control (DMSO); Figure 2 B is the result diagram of scheme 1; Figure 2 C is the result graph of Scheme 2; Figure 2 D is the result diagram of scheme 3; Figure 2 E is the result graph of Scheme 4; Figure 2 F is the result graph of Scheme 5; Figure 2 G is the result graph of Scheme 6; Figure 2 H is the result graph of Scheme 7; Figure 2 I is the result diagram of Scheme 8; Figure 2 J is the result diagram of Scheme 9);
[0047] Figure 3 : The drug sensitivity test results of Experiment 3 in Example 2 ( Figure 3 A is the result graph of the control (DMSO); Figure 3 B is the result diagram of scheme 1; Figure 3 C is the result graph of Scheme 2; Figure 3 D is the result diagram of scheme 3; Figure 3 E is the result graph of Scheme 4; Figure 3 F is the result graph of Scheme 5; Figure 3 G is the result graph of Scheme 6; Figure 3 H is the result graph of Scheme 7; Figure 3 I is the result diagram of Scheme 8; Figure 3 J is the result graph of Scheme 9; Figure 3 K is the result diagram of Scheme 10). DETAILED DESCRIPTION
[0048] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.
[0049] The following experimental materials and instruments were commercially available unless otherwise specified.
[0050] Example 1: Tumor organoid-immune cell co-culture
[0051] (1) Organoid digestion and counting
[0052] Observe the state of the 48-well plate organoids under a microscope. When there are 2,000 to 3,000 organoids in each protocol, co-culture with immune cells.
[0053] Discard the supernatant from the wells in the well plate where the experiment is to be conducted, add 200 μL of Tryple for digestion, and gently pipette with a 200 μL pipette until most of the organoids are in the form of microspheres;
[0054] After digestion was complete, 200 μL of washing solution was added to terminate the digestion;
[0055] Use a 200μL pipette to transfer the cell suspension in the 48-well plate to a 1.5ml EP tube;
[0056] Take 10 μL of single cell suspension and 10 μL of AOPI dye solution and mix them respectively, and then take 20 μL of the mixture to count with a cell counter.
[0057] (2) T cell expansion
[0058] In a 15 mL centrifuge tube, add Hank's Buffer equal to the volume of blood, then add 5 mL of blood sample and mix gently to obtain a mixed solution;
[0059] Add lymphocyte separation solution to a 50 mL centrifuge tube, and add the mixed solution in the above step dropwise onto the upper layer of lymphocyte separation solution along the wall once per second. The volume ratio of lymphocyte separation solution to blood sample is 2:1 (2:1 with blood sample, 1:1 with blood sample + Hank's buffer);
[0060] Centrifuge at 4°C, 2000 rpm, 20 min;
[0061] The PBMC layer was aspirated, washed with 1 mL PBS, centrifuged at 1500 rpm for 5 min at 4°C, repeated washing 3 times, and counted;
[0062] T cells were cultured in suspension using T cell culture medium (T cell culture medium + CD3 + CD28), expanded, and when the number of organoids was expanded to 10 times, they were co-cultured with the organoids and co-cultured in a carbon dioxide incubator at 37°C for 5 days (T cell seeding density was 2000-10000 / well).
[0063] (3) T cell count
[0064] Observe the status of T cells in the 48-well plate under a microscope to determine whether co-culture is possible;
[0065] Transfer the cell suspension in the wells to be tested in the 48-well plate to a 1.5 ml EP tube;
[0066] 10 μL of T cell suspension and AOPI were mixed respectively, and 20 μL of the mixture was counted using a cell counter.
[0067] (4) Calculation of cell suspension volume
[0068] Calculate the concentration of organoid cell suspension and T cell suspension based on the cell counting results;
[0069] Calculate the required volumes of organoid cell suspension and T cell suspension so that the ratio of T cells to organoid cells in each well is 10:1.
[0070] (5) Co-culture
[0071] According to the calculated volume, the organoid cell suspension and the T cell suspension were mixed, the number of organoid cells was 1000-1500 per well, and the number of T cells was 10000-15000 per well (organoids were 3D in morphology, and T cells were single-cell; T cell calculation method: first use a counter to measure the number of cells in 1 mL of suspension, and then use the cell suspension of the corresponding volume of the required cells; organoid counting method: digest the organoids for 5 minutes, stop digestion, centrifuge at 4°C, 1500 rpm for 5 minutes, and count using a cell counter).
[0072] Centrifugation, 1500 rpm, 5 min;
[0073] Take out the EP tube after centrifugation, discard the supernatant, and add the corresponding Matrigel and co-culture medium (the culture medium is half organoid culture medium and half T cell culture medium) according to the experimental protocol, with 10 μL of Matrigel per well;
[0074] Matrigel and 90 μL of cells were mixed and plated evenly in a 96-well plate;
[0075] Add corresponding drugs to the corresponding wells in the co-culture 96-well plate;
[0076] On Day 5, record the experimental results.
[0077] Example 2: Drug sensitivity experiment
[0078] The following uses colorectal cancer chemotherapy, targeted therapy, and immunotherapy as examples to illustrate that the drugs are added in "Step 5 Co-culture: Adding corresponding drugs to corresponding wells in a 96-well plate" in Example 1:
[0079] Scheme 1: Capecitabine alone;
[0080] Regimen 2: FOLFOX (5-fluorouracil, folinic acid (sensitizer), oxaliplatin: 25:5:1);
[0081] Regimen 3: FOLFIRI (5-fluorouracil, tetrahydrofolate (sensitizer), irinotecan: 2:2:1);
[0082] Regimen 4: FOLFOXIRI (5-fluorouracil, leucovorin (sensitizer), oxaliplatin, irinotecan: 2:2:2:1);
[0083] Option 5: FOLFOX + EGFR inhibitor (cetuximab: erbitux or panitumumab) must be wild-type KRAS exon 2 (25:5:1:1);
[0084] Regimen 6: FOLFOX + VEGFR inhibitor (bevacizumab; Avastin) KRAS gene mutation (25:5:1:1);
[0085] Regimen 7: pembrolizumab, nivolumab (PD-1 inhibitor) for dMMR or MSI-H (1:2000);
[0086] Regimen 8: trastuzumab (HER2 positive) (1:1000);
[0087] Regimen 9: Regorafenib (VEGFR, FGFR, PDGFR, KIT, BRAF, RET, etc. inhibitor) (1:2000);
[0088] Regimen 10: Vemurafenib (B-Raf V600E inhibitor) (1:2000).
[0089] This time, samples from three patients (CA012, CA015, and CA017) were screened for co-culture and drug sensitivity testing; the flow cytometry results are as follows:
[0090] Experiment 1: Patient CA012
[0091] Using DMSO as a control, the drug sensitivity experiments of the above schemes 1-8 were performed respectively. The results are as follows Figure 1 As shown;
[0092] Experiment 2: Patient CA015
[0093] Using DMSO as a control, the drug sensitivity experiments of the above schemes 1-9 were performed respectively. The results are as follows Figure 2 As shown;
[0094] Experiment 3: Patient CA017
[0095] Using DMSO as a control, the drug sensitivity experiments of the above schemes 1-10 were performed respectively. The results are as follows Figure 3 shown.
[0096] The above results show that the present application has discovered that by co-culturing organoids with T cells, flow cytometry is used to detect the sensitivity of different medication regimens and the lethality of different drugs to organoids, thereby reflecting the sensitivity of the patient's autologous tissue to the medication regimen.
[0097] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A method for co-culturing tumor organoids and immune cells, characterized in that: The following steps are involved: S1, Organoid digestion and counting; S2, T cell expansion; S3, T cell count; S4, calculation of cell suspension volume; S5, co-culture; S51, according to the volume calculated in step S4, the organoid cell suspension and the T cell suspension are mixed, the number of organoid cells is 1000-1500 per well, and the number of T cells is 10000-15000 per well; S52, centrifugation, 1500 rpm, 5 min; S53, discard the supernatant after centrifugation, add the corresponding Matrigel and co-culture medium according to the experimental protocol, and Matrigel is 10 μL per well; S54, Matrigel and 90 μL of cells were mixed and evenly seeded in 96-well plates for co-culture.
2. The method according to claim 1, characterized in that Step S1 includes the following steps: Observe the state of the 48-well plate organoids under a microscope. When there are 2,000 to 3,000 organoids in each protocol, co-culture with immune cells. Discard the supernatant from the wells in the well plate where the experiment is to be conducted, add 200 μL of Tryple for digestion, and gently pipette with a 200 μL pipette until most of the organoids are in the form of microspheres; After digestion was complete, 200 μL of washing solution was added to terminate the digestion; Use a 200μL pipette to transfer the cell suspension in the 48-well plate to a 1.5ml EP tube; Take 10 μL of single cell suspension and 10 μL of AOPI dye solution and mix them respectively, and then take 20 μL of the mixture to count with a cell counter.
3. The method according to claim 1, characterized in that Step S2 includes the following steps: In a 15 mL centrifuge tube, add Hank's Buffer equal to the volume of blood, add the blood sample, and mix gently to obtain a mixed solution; Add lymphocyte separation solution to a 50 mL centrifuge tube, and add the mixed solution in the above step dropwise onto the upper layer of lymphocyte separation solution along the wall one second at a time. The volume ratio of lymphocyte separation solution to blood sample is 2:
1. Centrifuge at 4°C, 2000 rpm, 20 min; The PBMC layer was aspirated, washed with 1 mL PBS, centrifuged at 1500 rpm for 5 min at 4°C, repeated washing 3 times, and counted; T cells were cultured in suspension using T cell culture medium and expanded. When the number of cells expanded to 10 times that of organoids, they were co-cultured with organoids in a carbon dioxide incubator at 37°C for 5 days.
4. The method according to claim 3, characterized in that The T cell seeding density was 2000-10000 / well.
5. The method according to claim 1, characterized in that Step S3 includes the following steps: Observe the status of T cells in the 48-well plate under a microscope to determine whether co-culture is possible; Transfer the cell suspension in the wells to be tested in the 48-well plate to a 1.5 ml EP tube; 10 μL of T cell suspension and AOPI were mixed respectively, and 20 μL of the mixture was counted using a cell counter.
6. The method according to claim 1, characterized in that Step S3 includes the following steps: Calculate the concentration of organoid cell suspension and T cell suspension based on the cell counting results; Calculate the required volumes of organoid cell suspension and T cell suspension so that the ratio of T cells to organoid cells in each well is 10:
1.
7. The method according to claim 1, characterized in that In step S53, the co-culture medium is half organoid medium and half T cell medium.
8. Use of the method according to any one of claims 1 to 7 in the preparation of a product for detecting a patient's sensitivity to medication.
9. The use according to claim 8, characterized in that: The products include detection kits.