A method for constructing a non-small cell lung cancer organoid and application thereof in screening of chemotherapeutic drug sensitivity

Abstract

The application discloses a construction method of a non-small cell lung cancer organoid and application of the non-small cell lung cancer organoid in chemotherapy drug sensitivity screening, and relates to the technical field of tumor model construction; the construction method comprises the following steps: taking human non-small cell lung cancer cells NCI-H1299, wherein the NCI-H1299 is derived from lymph node metastasis, p53 protein is lost and is not expressed. In the application, the human non-small cell lung cancer cell line NCI-H1299 is selected as seed cells to construct an organoid, and a standardized reference model is formed; compared with patient-derived primary tissues, the cell line has a consistent genetic background and can be stably passaged, and heterogeneity interference caused by individual differences is avoided; the obtained organoid is uniform in size, good in batch repeatability, and can be used as a standardized reference model in the field of NSCLC organoids, and is used for methodological verification of a drug screening platform, process quality control and data comparison between different laboratories.

Description

Technical Field

[0001] This invention relates to the field of tumor model construction technology, and in particular to a method for constructing organoids for non-small cell lung cancer and its application in screening for chemotherapeutic drug sensitivity. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer, accounting for approximately 85% of all lung cancer cases. Due to the insidious nature of early symptoms, most patients are diagnosed at an advanced stage, losing the opportunity for surgical cure. Platinum-based combination chemotherapy (such as cisplatin combined with pemetrexed, carboplatin combined with gemcitabine, etc.) is currently the standard first-line treatment for advanced NSCLC. However, due to tumor heterogeneity, different patients exhibit significant differences in sensitivity to the same chemotherapy regimen, often leading to unnecessary toxic side effects and poor efficacy for some patients. Therefore, establishing an in vitro model that can accurately predict chemotherapy drug sensitivity is of significant clinical importance for achieving individualized precision treatment of NSCLC.

[0003] Currently, antitumor drug screening mainly relies on traditional two-dimensional monolayer cell culture and patient-derived xenograft models. While 2D cell culture is simple and inexpensive, its lack of three-dimensional tissue structure, cell-cell interactions, and in vivo microenvironment leads to low accuracy in predicting in vivo drug responses, often resulting in in vitro sensitivity but in vivo ineffectiveness. Although PDX models can better preserve the molecular characteristics and heterogeneity of the primary tumor, their modeling cycle is long, success rate is low, cost is high, and they rely on immunodeficient mice, making it difficult to meet the needs of rapid clinical drug sensitivity testing.

[0004] In NSCLC organoid research, current technologies mostly use fresh tumor tissue surgically removed from patients for primary culture. However, this approach has several limitations: First, the source of primary tissue is affected by individual differences, resulting in strong genetic heterogeneity and significant batch-to-batch variations, making it difficult to use as a standardized reference model for methodological validation and quality control in drug screening platforms. Second, existing methods for constructing organoids using classic NSCLC cell lines such as NCI-H1299 lack standardized procedures, leading to ambiguity in key parameters such as cell seeding density, matrix gel ratio, and culture medium composition, resulting in inconsistent organoid sizes and poor reproducibility of drug screening data. Third, current drug screening assays largely rely solely on the CCK-8 assay to detect cell viability, lacking in-depth analysis of apoptosis mechanisms and hindering comprehensive evaluation of drug efficacy. Furthermore, the time from sample acquisition to successful construction of primary organoids is relatively long. In scenarios such as high-throughput initial screening of chemotherapy drugs and comparison of the efficacy of combination regimens, standardized, stably passaged cell-derived organoids are still needed as reference models. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for constructing organoids for non-small cell lung cancer and its application in screening for chemotherapeutic drug sensitivity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing organoids from non-small cell lung cancer includes the following steps: Step 1: Obtain human non-small cell lung cancer cells NCI-H1299, which are derived from lymph node metastases and lack p53 protein and do not express it; Step 2: NCI-H1299 cells were routinely adherent and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C and 5% CO2 saturated humidity. When the cell confluence reached 80%-90%, the cells were passaged at a ratio of 1:3, and the logarithmic phase cells were used for organoid construction. Step 3: Digest and collect NCI-H1299 cells, centrifuge and resuspend for counting, and aliquot into 2000 cells / well; Step 4: Discard the supernatant, resuspend the cells in organoid culture medium, mix the cell suspension with organoid matrix gel at a volume ratio of 1:3, and pipette evenly. Step 5: Take 35 μl of cell-Matrix gel mixture and drop it into the center of the bottom of the well of a 24-well plate to form a droplet. Invert the plate and place it in a 37°C, 5% CO2 incubator for 25 min to allow the matrix gel to solidify. Step 6: After confirming gel solidification, add 500 μl of non-small cell lung cancer organoid culture medium to each well and continue culturing in a 37°C, 5% CO2 incubator to obtain non-small cell lung cancer organoids.

[0007] Preferred method: The digestion in step 3 is as follows: Add 1-3 ml of 0.25% trypsin to a T25 culture flask, digest at 37°C for 2-5 min, and after observing under a microscope that the cells have shrunk and become rounded or a few have detached, gently shake the bottom of the flask to remove all the cells. Add complete culture medium containing 10% FBS to stop the digestion, and centrifuge at 1000 rpm to collect the precipitate.

[0008] Preferably, in step 4, the organoid matrix gel is thawed at 4°C until completely liquefied, the pipette tip is pre-cooled at 4°C, and the cell-matrix mixing operation is performed on ice to avoid the matrix gel from solidifying due to temperature rise.

[0009] Preferably, the non-small cell lung cancer organoid culture medium is based on DMEM / F12 and contains the following components: 1× penicillin antibody, 1× B27, 1× N2, 1× Glutamax, 1mM NAC, 50-100ng / ml Wnt-3A, 50ng / ml EGF, 50-100ng / ml FGF10, 5-10μMY-27632, 5-10μMSB202190; preferably, it also contains 100ng / ml Noggin.

[0010] Preferred method: Change the medium every 2-4 days during organoid culture: carefully remove the original culture medium and add 500 μl of fresh organoid culture medium along the bottom of the well plate; when passage the organoids, add 300 μl of trypsin to each well to disperse the gel droplets, digest at 37°C for 5-10 min until the organoids are digested into small cell clusters but not completely into single cells, terminate with serum-containing culture medium, collect the precipitate by centrifugation, and passage at a ratio of 1:3. Add the cell-Matrix gel mixture at 10 μl / well or 38 μl / well to a new 24-well plate, invert for 25 min to solidify, and then add culture medium to continue culturing.

[0011] Application of non-small cell lung cancer organoids obtained using the above methods in screening for chemotherapy drug sensitivity.

[0012] Preferably, the chemotherapy regimen used for the chemotherapy drug sensitivity screening is selected from one of the following three combination regimens: (a) Cisplatin + Pemetrexed; (b) Cisplatin + Dorcetaxel; (c) Carboplatin + Gemcitabine;

[0013] The cisplatin, carboplatin, and gemcitabine stock solutions were prepared with ultrapure water or DMF, and the docetaxel and pemetrexed stock solutions were prepared with DMSO. The concentration of each stock solution was 20 mM. After sterilization by 0.22 μm filter membrane, the solutions were aliquoted and stored at -20℃. When using, the solutions were diluted with organoid culture medium: 1 μl of cisplatin stock solution + 1 μl of pemetrexed stock solution, or 1 μl of cisplatin stock solution + 1 μl of docetaxel stock solution, or 1 μl of carboplatin stock solution + 1 μl of gemcitabine stock solution were added to every 2 ml of organoid culture medium.

[0014] Preferred method: Drug treatment steps are as follows: Select organoid wells that are in good condition and uniform in size, remove the original culture medium, add 500 μl of drug-containing culture medium per well, continue culturing, and then detect.

[0015] Preferred method: The effect of chemotherapy drugs on organoid proliferation was detected using the CCK-8 assay. The specific steps are as follows: (1) After drug treatment, the organoids were digested into single cells, collected by centrifugation, resuspended in 300 μl of organoid culture medium, and transferred to 96-well plates, 100 μl / well; (2) Add 10 μl of CCK-8 solution to each well and incubate at 37°C in the dark for 2 h; (3) The absorbance OD value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader; (4) Calculate cell viability and inhibition rate using the following formulas: Cell viability (%) = [A(experimental group) − A(blank group)] / [A(control group) − A(blank group)] × 100%; Cell inhibition rate (%) = 1 − cell survival rate; In this group, A (blank group) consists of wells containing only culture medium + CCK-8, A (control group) consists of wells containing organoids without the drug + CCK-8, and A (experimental group) consists of wells containing organoids with the drug + CCK-8.

[0016] Preferred method: Annexin V-FITC / PI double staining flow cytometry was used to detect organoid apoptosis. The specific steps are as follows: (1) After drug treatment, the organoids were digested into single cells and the precipitate was collected by centrifugation; (2) Resuspend in Annexin V-FITC binding buffer, centrifuge at 2000 rpm for 5 min, discard the supernatant, and resuspend in 185 μl of Annexin V-FITC binding buffer; (3) Add 5 μl Annexin V-FITC and incubate at room temperature in the dark for 10 min; (4) Add 10 μl of PI staining solution and place in an ice bath away from light; (5) Flow cytometry detection, with Annexin V-FITC green fluorescence and PI red fluorescence to distinguish quadrants: Q1-LR is early apoptosis, Q1-UR is late apoptosis, Q1-UL is necrosis, and Q1-LL is live cells; apoptosis rate = Q1-UR + Q1-LR.

[0017] The beneficial effects of this invention are as follows: 1. This invention uses the NCI-H1299 et al. non-small cell lung cancer cell line as seed cells to construct organoids, forming a standardized reference model. Compared with patient-derived primary tissues, the cell line has a consistent genetic background and can be stably passaged, avoiding heterogeneity interference caused by individual differences. The resulting organoids are uniform in size and have good batch reproducibility. It can be used as a standardized reference model in the field of NSCLC organoids for methodological validation, process quality control, and data comparison between different laboratories in drug screening platforms. At the same time, this standardized model can also serve as a reference for the development of primary organoid construction processes, providing reproducible parameter benchmarks for subsequent expansion to patient-derived samples, and has clear industrial application value.

[0018] 2. This invention not only verifies the applicability of commercial culture medium (culture medium 3), but also provides two self-prepared culture medium formulations (culture medium 1 and 2). For culture medium 2, by introducing Noggin, high concentrations of Wnt-3A, FGF10, and specific concentrations of Y-27632 (ROCK inhibitor) and SB202190 (p38 inhibitor), the dryness maintenance and survival of NSCLC organoids are effectively promoted, solving the problems of easy differentiation and low survival rate of NSCLC organoids in traditional culture media.

[0019] 3. This invention establishes a dual evaluation system, in which CCK-8 quantitatively reflects the number of live cells through 450nm absorbance and calculates the inhibition rate; Annexin V-FITC / PI dual staining accurately distinguishes between early apoptosis, late apoptosis, and necrotic cells; the combination of the two systems comprehensively evaluates the efficacy of chemotherapy drugs from two dimensions: the reduction in number and the mechanism of cell death, resulting in more detailed and reliable data.

[0020] 4. This invention clarifies the specific solvent (DMF or DMSO), concentration, and filtration sterilization method for preparing the mother liquor; it provides a specific dilution gradient, which not only ensures the solubility of the drug in the culture medium, but also simulates the combination drug use logic of clinical chemotherapy drugs (cisplatin / carboplatin + third-generation chemotherapy drugs), and has high clinical translation reference value. Attached Figure Description

[0021] Figure 1 These are images of the revived cell state and tumor organoids observed under a microscope in this invention. Figure 2 This is a cell photograph used in the CCK8 assay to detect the effect of drugs on organoid proliferation in this invention. Figure 3 This is a schematic diagram illustrating the principle of flow cytometry apoptosis detection in this invention. Figure 4 This is a graph showing the detection results of the blank control group in this invention; Figure 5 This is a graph showing the detection results of the normal culture group in this invention; Figure 6 This is a graph showing the detection results of drug treatment group 1 in this invention; Figure 7 The image shows the detection results of drug treatment group 2 in this invention; Figure 8 This is a graph showing the detection results of drug treatment group 3 in this invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1: In this example, the NCI-H1299 human non-small cell lung cancer cell line was used as the seed cell to construct a standardized reference model of NSCLC organoids, which was used to verify the stability and repeatability of the construction process described in this patent, as well as the standardized implementation of the chemotherapy drug sensitivity detection process.

[0024] 1.1 Cell Procurement

[0025] NCI-H1299, human non-small cell lung cancer cells.

[0026] Cell Description: The cells originated from a single lymph node metastasis, uniformly partially lacking p53 protein and exhibiting no p53 protein expression. NCI-H1299 cells can synthesize NMB protein at 0.1 pmol / mg protein, but do not synthesize gastrin-releasing peptide (GRP).

[0027] Growth morphology: adherent growth, epithelial cell-like, polygonal.

[0028] Culture conditions: Culture the cells in RPMI-1640 medium containing 10% high-quality fetal bovine serum and 1% penicillin-streptomycin antibiotics. Change the medium every 2-3 days. Passage the cells when they reach 80% confluence. The passage ratio can be 1:3. Culture the cells in an incubator at 37°C, 5% CO2 and saturated humidity.

[0029] 1.2 NCI-H1299 cells arrived

[0030] After the cells arrive, follow the instructions to place them in an incubator for 2-3 hours, remove the culture medium from the culture flask, and replace it with fresh culture medium.

[0031] Preparation of NCI-H1299 normal growth culture medium: Take 45ml of RPMI-1640 medium, add 5ml of fetal bovine serum, and finally add 0.5ml of penicillin-streptomycin stock solution to make the final concentrations of penicillin and streptomycin 100U / mL and 100ug / mL, respectively. Store at 4℃ and warm to room temperature before use.

[0032] Aseptic technique precautions: When culturing cells, it is essential to maintain a sterile and clean work area. First, sterilize with UV lamp and ozone for 1 hour, then ventilate for 30 minutes. Before operation, change into cell culture room slippers, put on disposable rubber gloves and disinfect your hands with 75% ethanol, put on a lab coat, and wear a disposable mask and cap. The entire aseptic operation should be carried out around the alcohol lamp.

[0033] 1.3 Cell passage

[0034] Cell passage method: Observe the culture flask under a microscope until 80%-90% confluence. Perform the operation on a clean bench. First, remove the old culture medium from the culture flask and wash with PBS 2-3 times. Add about 1-3 mL of trypsin digestion solution to the T25 culture flask and digest it according to this ratio. Shake to spread the digestion solution evenly and incubate at 37 degrees Celsius for about 2-5 minutes. After observing the cells shrinking and becoming round or a few detaching under the microscope, gently shake the bottom of the flask to remove all the cells. Add 2-3 mL of complete culture medium and gently pipette to collect the cells into a centrifuge tube. Discard the supernatant, add culture medium, and gently pipette to form a cell suspension. Pass the cells into sterile culture flasks at a ratio of 1:3, add culture medium, and continue culturing or experimenting.

[0035] Cell density: 80%, passage ratio 1:3, from 1 T25 cell to 3 T25 flasks.

[0036] 1.4 Cell cryopreservation and passage

[0037] Once the new cells arrive, the seed cells should be cryopreserved as soon as possible. Cryopreservation method: The principle is slow freezing: Place the cells at 4°C for 30 minutes, then transfer them to -20°C for 2 hours, followed by overnight freezing at -80°C. Finally, store them in liquid nitrogen. Alternatively, use a programmed freezing box to freeze them overnight at -80°C, and then store them in liquid nitrogen the next day. Select cells in the logarithmic growth phase, preferably those that have been passaged 2-3 times.

[0038] First, digest the cells with 0.25% trypsin. Collect the digested cells in centrifuge tubes and count them. Centrifuge at 1000 rpm and discard the supernatant. Resuspend the cells in cell freezing medium and aliquot them into cryovials at 1 mL per tube. Cryopreserve the cells according to the slow freezing principle described above.

[0039] Preparation of cryopreservation solution: Add 5% DMSO to the culture medium containing 20% ​​fetal bovine serum, mix well, and store at 4°C.

[0040] Cell quantity: 3 bottles of T25, of which 2 bottles were used for seed cell cryopreservation, for a total of 2 vials cryopreserved; The other bottle was used to continue the culture. After subculturing at a ratio of 1:3, the culture was inoculated into three T25 bottles and placed in an incubator for further culture.

[0041] 1.5 Secondary cell cryopreservation and passage

[0042] There are a total of 3 bottles of cells. The same procedure as before is followed. Two bottles are used for cell cryopreservation, and a total of 2 vials are cryopreserved. The other bottle was used to continue the culture. After subculturing at a ratio of 1:3, the culture was inoculated into three T25 bottles and placed in an incubator for further culture.

[0043] 1.6 Third cell cryopreservation and tumor organoid culture

[0044] Three vials of cells were prepared, and the same procedures were followed as before. Two vials were used for cell cryopreservation, resulting in a total of two vials cryopreserved. A total of six vials were cryopreserved at this point, and no further cryopreservation was carried out.

[0045] Another bottle of cells was used for tumor organoid culture.

[0046] The tumor organoid culture medium is prepared as follows:

[0047] Preparation methods for each reagent: 1. B27, N2, Glutamax, and dual antibodies: These are the original solutions; add them directly according to the specified ratio. 2. Nac: Molecular weight 163.2. Weigh 100mg of solid powder, add 1.225ml of PBS, and dissolve to obtain a 500mM stock solution; 3. Noggin: 20ug, directly added to 0.200ml PBS, dissolved to form a stock solution of 100ug / ml; 4. Wnt-3A: 25ug, directly added to 0.250ml PBS, to dissolve into a stock solution of 100ug / ml; 5. EGF: 20ug, directly added to 0.400ml PBS, dissolved to form a stock solution of 50ug / ml; 6. FGF10: 20ug, directly added to 0.400ml PBS, to dissolve into a stock solution of 50ug / ml; 7. Y-27632: molecular weight 247.34, 1 mg, directly added to 4.043 ml PBS, dissolved to form a 1 mM stock solution; 8. SB202190: Molecular weight 331.34, 5mg, directly added to 1.509ml DMSO, dissolved to form a 10mM mother liquor; After each reagent is prepared, it is dispensed into smaller portions and stored at -20°C.

[0048] Tumor organoid culture methods: 1. Thaw the organoid matrix gel at 4°C in advance, ensuring it is fully thawed; pre-cool the pipette tip in a refrigerator.

[0049] 2. After digesting the cells using standard methods, centrifuge to collect the precipitate, resuspend it in culture medium, and then count the cells. 3. Divide the cells into three centrifuge tubes at a ratio of 2000 cells per well, and centrifuge again; 4. Remove the supernatant and resuspend the cells in three different organ culture media, with a culture medium volume of 10 μl / well. 5. Take out the organoid-specific matrix gel that has been pre-cooled on ice, and add matrix gel to the cell suspension at a volume ratio of 1:3 (do this as quickly as possible to avoid the matrix gel from solidifying due to temperature rise), and mix well by pipetting.

[0050] 6. Immediately take 35 μL of suspension and carefully drop it into the center of the 24-well plate to form a droplet. Then invert the culture plate and place it in an incubator for 25 minutes to allow the substrate gel to solidify. 7. After the gel droplets solidify, carefully add 500 μL of organoid culture medium to each well and return the well to the incubator for further culture.

[0051] 8. The remaining cells were seeded into T25 flasks at a ratio of 1:3 and cultured using standard methods.

[0052] 2.1 Cell resuscitation

[0053] 1. Resuscitate frozen seed cells.

[0054] 2. Adhere to the principle of rapid thawing. First, adjust the water bath to 37°C. Take the cryovial out of the liquid nitrogen tank and quickly immerse it in 37°C warm water. Gently shake it to allow the contents to thaw quickly (preferably within 1 minute). 3. Remove the cryovial, gently shake it up and down, and then open it after sterilizing it with 75% alcohol;

[0055] 4. Use a pipette to aspirate the dissolved cell suspension, inject it into a centrifuge tube, and add 5 mL of cell culture medium containing 10% fetal bovine serum (FBS).

[0056] 5. Centrifuge at 1000 rpm for 5 min and remove the supernatant; 6. Add 1 mL of culture medium and pipette to form a suspension. Dilute appropriately with cell culture medium containing 10% FBS, then inoculate into culture flasks and incubate at 37°C, saturated humidity, and 5% CO2. 7. Change the culture medium after 24 hours. Then, change the culture medium again depending on the cell growth and the color of the culture medium (generally wait until the culture medium turns yellow) and continue culturing.

[0057] 2.2 Viewing the status of revived cells

[0058] like Figure 1 (A) The cells are in good condition and can be passaged. The next generation of cells after passage can be used for organoid culture.

[0059] The cell passage ratio is 1:3. Take 1 / 3 of the cells from the T25 culture flask, centrifuge and resuspend the cells, then seed them into a new T25 culture flask and place it in an incubator for further culture.

[0060] 2.3 Tumor organoid culture

[0061] Cells from T25 culture flasks were used to culture tumor organoids, using the same method as before.

[0062] Note: Minimize cell digestion time, rewarm the cell culture medium beforehand, and resuspend the cells in the culture medium during the counting process after centrifugation before storing them in an incubator.

[0063] Inoculation volume: 2000 cells / well, 35 μL / well, culture medium: matrix gel = 1:3.

[0064] After gel inoculation, invert the plate in an incubator for 25 minutes, then gently shake the plate to confirm that the gel has completely solidified.

[0065] Add 500 μl of various organoid culture media to each well.

[0066] Passage of remaining cells: passage at a ratio of 1:3, retaining the cell count of one T25 flask.

[0067] 2.4 Observation of Tumor Organoids

[0068] Observe under a microscope, such as Figure 1 (B) The cells were found to be in good condition, with cell spheroids already visible. The culture was continued.

[0069] 2.5 Observation of Tumor Organoids

[0070] Microscopic observation revealed that the tumor organoid spheroids were growing larger, and the state of the three culture media was not significantly different.

[0071] Culture medium replacement: Carefully remove the original culture medium, and then carefully add 500 μl of fresh culture medium along the bottom of the well plate.

[0072] Organoid culture medium 1, such as Figure 1 (C); Organoid culture medium 2, such as Figure 1 (D); Organoid culture medium 3, such as Figure 1 (E), Commercial complete culture medium.

[0073] 2.6 Observation of Tumor Organoids

[0074] Microscopic observation revealed that the tumor organoid spheroids were growing. Among them, the organoids cultured in organoid culture medium 3 (commercial lung cancer tumor organoids) were relatively better, and this culture medium will be selected for subsequent processing.

[0075] Organoid culture medium 1, such as Figure 1 (F); Organoid culture medium 2, such as Figure 1 (G); Organoid culture medium 3, such as Figure 1 (H), commercialized lung cancer tumor organoids.

[0076] Organoid passage: 1. First remove the original culture medium, then carefully wash twice with PBS; 2. Add 300 μl of trypsin to each well and use a pipette tip to disperse the organoid gel; 3. Place in an incubator for 5-10 minutes to digest, observing the degree of organoid digestion multiple times during this period; 4. When most of the organoids have been digested into small cell clusters, but not completely into single cells, stop the digestion with 500 μl of serum-containing culture medium. 5. Centrifuge to collect the precipitate and wash once with organoid culture medium; 6. After collecting the precipitate, passage it at a ratio of 1:3, and add commercial organoid complete culture medium at a volume of 10 μl / well of cells. 7. Add 3 times the volume of matrix gel, carefully resuspend the cells, mix as thoroughly as possible and minimize the formation of air bubbles; 8. Add the gel to a new 24-well plate at a volume of 38 μl / well and incubate it upside down in an incubator for 25 min; 9. Gently shake the plate to confirm that the gel has completely solidified, and add 500 μl of various organoid culture media to each well.

[0077] 2.7 Observation and fluid exchange of tumor organoids

[0078] Observing organoids under a microscope, such as Figure 1 (I) It can be seen that it grows into a relatively uniform and round sphere, and is in good condition.

[0079] Change medium: Carefully remove the original culture medium and then carefully add 500 μl of fresh culture medium along the bottom of the well plate.

[0080] 2.8 Observation and passage of tumor organoids

[0081] Observing organoids under a microscope, such as Figure 1 (J) is in good condition, with an increase in tumor spheroids and a faster rate of culture medium consumption, and can be passaged.

[0082] Subculturing method: As described above, subculture at a ratio of 1:3 to expand the culture.

[0083] 2.9 Organoid Observation and Fluid Change

[0084] Observing organoids under a microscope, such as Figure 1 (K), in good condition, is a sphere of varying sizes.

[0085] Medium change: Carefully remove the original culture medium, and then carefully add 500 μl of fresh culture medium along the bottom of the well plate.

[0086] 2.10 Organ cryopreservation and passage

[0087] Cryopreservation: First, digest and collect the precipitate of organoids according to the organoid passage method, then resuspend the cells in cell cryopreservation solution and aliquot them into cryovials at 1 mL per tube. Cryopreserve the cells as described above.

[0088] Passage: The organoids are passaged at a ratio of 1:3, and the passaged organoids are used for drug experiments.

[0089] 2.11 Organoid fluid exchange

[0090] Change medium: Carefully remove the original culture medium and then carefully add 500 μl of fresh culture medium along the bottom of the well plate.

[0091] 2.12 Organoid CCK8 Detection Plates

[0092] Grouping:

[0093] 1. Normal culture group

[0094] 2. Drug treatment group 1 (cisplatin + pemetrexed)

[0095] 3. Drug treatment group 2 (cisplatin + docetaxel)

[0096] 4. Drug treatment group 3 (carboplatin + gemcitabine)

[0097] Each group has 3 duplicate holes. To ensure the accuracy of the board laying, lay a few more holes, for a total of 20 holes (5 groups * 4 duplicate holes).

[0098] The procedure for passage of similar organs is consistent: Normally cultured lung cancer tumor organoids were digested and seeded into 24-well plates. After solidification by inverting the plates, culture medium was added. The remaining organoids were passaged for use in subsequent experiments.

[0099] 2.13 Organoid Drug Administration

[0100] The drug information is as follows:

[0101]

[0102] Drug preparation: (1) Cisplatin: MW=300.05, solid 5mg, +0.833ml DMF, →20mM stock solution.

[0103] (2) Pemetrexed disodium salt: MW=471.37, solid 5mg, +0.503ml ultrapure water, → 20mM stock solution, sterilized by 0.22μm filter.

[0104] (3) Docetaxel: MW=807.88, solid 25mg, +1.547mlDMSO, → 20mM stock solution.

[0105] (4) Carboplatin: MW=371.25, 10mg solid, +1.347ml DMSO, → 20mM mother liquor.

[0106] (5) Gemcitabine: MW=263.2, 10mg solid, +1.900ml ultrapure water, → 20mM stock solution, sterilized by 0.22μm filter.

[0107] After the above drugs are prepared, they are dispensed and stored at -29℃ and diluted with organoid culture medium.

[0108] dilution: Drug 1: 2 ml organoid culture medium + 1 μl cisplatin stock solution + 1 μl pemetrexed stock solution

[0109] Drug 2: 2 ml culture medium + 1 μl cisplatin stock solution + 1 μl docetaxel stock solution

[0110] Drug 3: 2 ml culture medium + 1 μl carboplatin stock solution + 1 μl gemcitabine stock solution

[0111] Organoid drug administration: 1. Observe that the organoids are in good condition and of appropriate size, and select uniform wells as test wells; 2. Carefully remove the original culture medium and add 1 / 2 / 3 of the drug to each well, 500 μl each; 3. Return to the incubator and continue culturing.

[0112] 3.1 CCK8 assay to detect the effect of drugs on organoid proliferation

[0113] CCK8 principle: The Cell Counting Kit (CCK-8) is a highly sensitive and rapid assay kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt), widely used for the detection of cell proliferation and cytotoxicity. WST-8 is an upgraded product of MTT. Its working principle is as follows: in the presence of an electron coupling reagent, it is reduced by mitochondrial dehydrogenases to generate a highly water-soluble orange-yellow formazan product. The intensity of the color after the reaction is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at 450 nm using a microplate reader, indirectly reflecting the number of viable cells. The CCK method has a wide range of applications, such as drug screening, cell proliferation, cytotoxicity, tumor drug sensitivity testing, and cytokine activity detection.

[0114] Required instrument: Multifunctional microplate reader (Varioskan LUX; ThermoFisher, USA)

[0115] Reagent: CCK8 assay kit (C0039; Beyotime, China)

[0116] Operating steps: 1. Observe the drug-treated organoids under a microscope and take pictures; 2. After the drug treatment is completed, the organoids are digested into single cells according to the organoid digestion method; 3. Centrifuge to collect the cell pellet, resuspend the cells in 300 μL of culture medium, and transfer 100 μL to each well of a 96-well plate.

[0117] 4. Add 10 μL of LCK-8 solution to each well.

[0118] 5. Place the culture plate in an incubator and incubate for 2 hours.

[0119] 6. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0120] Using the ELISA reader: Turn on the ELISA reader 10 minutes in advance to preheat the instrument. After the incubation is complete, open the software, set the detection parameters, then remove the caps from the well plates and place them on the stage. Click "Start" to begin the measurement.

[0121] Vitality calculation:

[0122] Cell viability (%) = [A(experimental group) - A(blank group)] / [A(control group) - A(blank group)] × 100%;

[0123] Cell inhibition rate (%) = [A (control group) - A (experimental group)] / A (control group) = 1 - cell survival rate; A (Experimental Group): Absorbance values ​​of treated cells and CCK8 solution; A (Blank group): Absorbance values ​​of wells containing culture medium and CCK8 solution but without cells; A (Control Group): Contains untreated cells and absorbance values ​​of CCK8 solution.

[0124] The CCK8 test results are as follows:

[0125] Cell photography: 1. Normal culture group, such as Figure 2 As shown in (A); 2. Drug treatment group 1, such as Figure 2 As shown in (B); 3. Drug treatment group 2, such as Figure 2 As shown in (C); 4. Drug treatment group 3, such as Figure 2 As shown in (D); Tumor organoid passage and plating:

[0126] CCK8 assay showed that the drug had an inhibitory effect on the proliferation of tumor organoids, so we proceeded directly to flow cytometry apoptosis assay.

[0127] Organoid passage: Passage is carried out using conventional methods.

[0128] 3.2 Flow cytometry apoptosis detection plate

[0129] Grouping: 1. Normal culture group; 2. Drug treatment group 1 (cisplatin + pemetrexed); 3. Drug treatment group 2 (cisplatin + docetaxel); 4. Drug treatment group 3 (carboplatin + gemcitabine); Each group has 3 duplicate holes. To ensure the accuracy of the board laying, lay a few more holes, for a total of 20 holes (5 groups * 4 duplicate holes). The procedure for passage of similar organs is consistent: Normally cultured lung cancer tumor organoids were digested and seeded into 24-well plates. After solidification by inverting the plates, culture medium was added. The remaining organoids were passaged for use in subsequent experiments.

[0130] 3.3 Organoid Drug Administration

[0131] Drug preparation: Take the drug stock solution stored at -20℃, thaw it at 4℃ for 1 hour, and then thaw it at room temperature for 15 minutes until it is in liquid state.

[0132] dilution: Drug 1: 2 ml organoid culture medium + 1 μl cisplatin stock solution + 1 μl pemetrexed stock solution; Drug 2: 2 ml culture medium + 1 μl cisplatin stock solution + 1 μl docetaxel stock solution; Drug 3: 2 ml culture medium + 1 μl carboplatin stock solution + 1 μl gemcitabine stock solution; Organoid drug administration: 1. Observe that the organoids are in good condition and of appropriate size, and select uniform wells as test wells; 2. Carefully remove the original culture medium and add 1 / 2 / 3 of the drug to each well, 500 μl each; 3. Return to the incubator and continue culturing.

[0133] 3.4 Flow cytometry detection of organoid apoptosis

[0134] Flow cytometer: CytoFLEX, Beckman, USA

[0135] A flow cytometer is a device that can detect and count cells in the form of a single-cell liquid stream passing through a laser beam. Because the cell marker tracer used in the detection is a fluorescent label, the flow cytometer used to separate and identify cells is also known as a fluorescence activated cell sorter (FACS), which is a powerful tool for separating and identifying cell populations and subpopulations.

[0136] The principle is as follows: a specific fluorescently labeled monoclonal antibody targeting a specific target cell surface molecule is added to a mixed cell population. This specific monoclonal antibody binds to its corresponding antigen target molecule, and the bound fluorescently labeled antibody remains on the surface of the specific cell, called a fluorescently labeled target cell. The mixed cell population containing the labeled cells is suspended in a certain volume of loading buffer, and then passed through the sample pipette well of the FACS instrument. The instrument will then form a micro-stream of single cells arranged in a single-cell configuration. When each cell is irradiated by the instrument's laser beam, the fluorescence on the cell is activated by the corresponding laser beam and emits corresponding fluorescence. The fluorescence emitted from the cell surface can be detected by a sensitive photomultiplier tube. The measured scattered light provides information on cell size and particle state; while the fluorescence emission intensity provides information on the antibody bound to the cell, thus reflecting the expression status of the corresponding molecule on the cell surface.

[0137] Flow cytometry apoptosis detection principle: Apoptosis is a fundamental characteristic of cells, playing a crucial role in embryonic development, tissue repair, and homeostasis. Annexin V is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 kDa that binds specifically and with high affinity to phosphatidylserine (PS), which is flipped out of the cell membrane during apoptosis. Using FITC-labeled Annexin V as a fluorescent probe, the occurrence of apoptosis can be detected by flow cytometry or fluorescence microscopy.

[0138] Propidium iodide (PI) is a nucleic acid dye that cannot pass through intact cell membranes. However, in late-stage apoptosis and dead cells, PI can pass through the cell membrane and stain the cell nucleus red. Using Annexin V in combination with PI can distinguish between early-stage apoptosis, late-stage apoptosis, and dead cells.

[0139] Annexin V-FITC flow cytometry apoptosis kit (AP101; Linko Biotech, China)

[0140] Operating steps: 1. Collect organoids after drug treatment and digest them into single cells; 2. Centrifuge to collect the cell pellet, gently resuspend the cells in Annexin V-FITC binding buffer, and take a small amount as a blank control.

[0141] 3. Centrifuge at 2000 rpm for 5 min, discard the supernatant, and gently resuspend the cells in 185 μl Annexin V-FITC binding solution.

[0142] 4. Add 5 μl Annexin V-FITC and mix gently.

[0143] 5. Incubate at room temperature in the dark for 10 minutes.

[0144] 6. Add 10 μl of propidium iodide staining solution, mix gently, and place in an ice bath away from light.

[0145] 7. Flow cytometry was then performed. Annexin V-FITC showed green fluorescence, and PI showed red fluorescence.

[0146] 4.1 Test Results:

[0147] Result Analysis: Meaning of the Four Quadrants

[0148] Q1-UL: Necrotic cells; Q1-UR: Late apoptotic cells; Q1-LR: Early apoptotic cells; Q1-LL: Cells that have not undergone apoptosis.

[0149] Calculate the apoptosis rate by selecting the sum of the Q1-UR and Q1-LR quadrant percentages;

[0150] Kb: Undyed blank control, used to distinguish between autofluorescence and specific fluorescence of intracellular substances, such as... Figure 4 As shown.

[0151] 1. Normal culture group, such as Figure 5 As shown; 2. Drug treatment group 1, such as Figure 6 As shown; 3. Drug treatment group 2, such as Figure 7 As shown; 4. Drug treatment group 3, such as Figure 8 As shown.

[0152] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing organoids for non-small cell lung cancer, characterized in that, Includes the following steps: Step 1: Obtain human non-small cell lung cancer seed cells, wherein the human non-small cell lung cancer seed cells include human non-small cell lung cancer cell lines; Step 2: The seed cells were conventionally adherent and cultured in RPMI-1640 medium containing fetal bovine serum and penicillin-streptomycin antibiotics at 37°C and 5% CO2 saturated humidity. When the cell confluence reached 80%-90%, the cells were passaged and the logarithmic phase cells were used for organoid construction. Step 3: Digest and collect seed cells, centrifuge and resuspend for counting, and dispense; Step 4: Discard the supernatant, resuspend the cells in organoid culture medium, mix the cell suspension with organoid matrix gel at a volume ratio of 1:3, and pipette evenly. Step 5: Add a drop of the cell-Matrix gel mixture to the center of the bottom of the well of the plate to form a droplet, then invert the plate and place it in a 37°C, 5% CO2 incubator to allow the matrix gel to solidify. Step 6: After confirming gel solidification, add non-small cell lung cancer organoid culture medium to each well and continue culturing to obtain non-small cell lung cancer organoids.

2. The construction method according to claim 1, characterized in that, In step 1, the human non-small cell lung cancer cell line is the NCI-H1299 cell line, which is derived from lymph node metastasis and has p53 protein deficiency and non-expression.

3. The construction method according to claim 1, characterized in that, In step 4, the organoid matrix gel is thawed at 4°C until completely liquefied, the pipette tip is pre-cooled at 4°C, and the cell-matrix mixing operation is carried out on ice to avoid the matrix gel from solidifying due to temperature rise.

4. The construction method according to claim 1, characterized in that, The non-small cell lung cancer organoid culture medium is based on DMEM / F12 and contains the following components: 1× penicillin antibody, 1× B27, 1× N2, 1× Glutamax, 1mM NAC, 50-100ng / ml Wnt-3A, 50ng / ml EGF, 50-100ng / ml FGF10, 5-10μMY-27632, and 5-10μMSB202190.

5. The construction method according to claim 1, characterized in that, The medium replacement method during organoid culture is as follows: carefully remove the original culture medium and add fresh organoid culture medium along the bottom of the well plate; when passage the organoids, add trypsin to each well to disperse the gel droplets, and digest until the organoids are digested into small cell clusters but not completely into single cells, and stop with serum-containing culture medium. After centrifugation to collect the precipitate, passage it in proportion, drop the cell-Matrix gel mixture onto a new well plate, invert it to solidify, add culture medium and continue culturing.

6. The use of the non-small cell lung cancer organoids obtained according to claim 1 in any of the following aspects: (a) Constructing a screening model for chemotherapy drugs in non-small cell lung cancer; (b) Preparation of a chemotherapeutic drug sensitivity assay kit for non-small cell lung cancer; (c) Evaluate the efficacy of chemotherapy drugs or combination chemotherapy regimens for non-small cell lung cancer.

7. The application according to claim 6, characterized in that, The chemotherapy regimen used for the chemotherapy drug sensitivity screening is selected from one of the following three combination regimens: (a) Cisplatin + Pemetrexed; (b) Cisplatin + Dorcetaxel; (c) Carboplatin + Gemcitabine; The cisplatin, carboplatin, and gemcitabine stock solutions were prepared with ultrapure water or DMF, while the docetaxel and pemetrexed stock solutions were prepared with DMSO. After sterilization by filtration membrane, the solutions were dispensed and stored separately.

8. The application according to claim 7, characterized in that, The drug processing steps are as follows: select organoid wells that are in good condition and uniform in size, remove the original culture medium, add drug-containing culture medium, continue culturing, and then test.

9. The application according to claim 7, characterized in that, The CCK-8 assay was used to detect the effect of chemotherapy drugs on organoid proliferation. The specific steps were as follows: (1) After drug treatment, the organoids were digested into single cells, collected by centrifugation, resuspended in 300 μl of organoid culture medium, and transferred to 96-well plates, 100 μl / well; (2) Add 10 μl of CCK-8 solution to each well and incubate at 37°C in the dark for 2 h; (3) The absorbance OD value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader; (4) Calculate cell viability and inhibition rate using the following formulas: Cell viability (%) = [A(experimental group) − A(blank group)] / [A(control group) − A(blank group)] × 100%; Cell inhibition rate (%) = 1 − cell survival rate; In this group, A (blank group) consists of wells containing only culture medium + CCK-8, A (control group) consists of wells containing organoids without the drug + CCK-8, and A (experimental group) consists of wells containing organoids with the drug + CCK-8.

10. The application according to claim 7, characterized in that, Annexin V-FITC / PI double staining flow cytometry was used to detect organoid apoptosis. The specific steps were as follows: (1) After drug treatment, the organoids were digested into single cells and the precipitate was collected by centrifugation; (2) Resuspend in Annexin V-FITC binding buffer, centrifuge at 2000 rpm for 5 min, discard the supernatant, and resuspend in 185 μl of Annexin V-FITC binding buffer; (3) Add 5 μl Annexin V-FITC and incubate at room temperature in the dark for 10 min; (4) Add 10 μl of PI staining solution and place in an ice bath away from light; (5) Flow cytometry detection, with Annexin V-FITC green fluorescence and PI red fluorescence to distinguish quadrants: Q1-LR is early apoptosis, Q1-UR is late apoptosis, Q1-UL is necrosis, and Q1-LL is live cells; apoptosis rate = Q1-UR + Q1-LR.

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